Method of selecting agents for the treatment of pain using neuronal differentiation

ABSTRACT

The present invention is based on the seminal concept of treating pain by promoting neuronal differentiation. The invention provides a screening method for the selection of agents for the purpose of treating pain, utilizing neuronal differentiation as selection criteria. Methods and libraries available for screening for the selection of candidate agents are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to U.S. Provisional Application Ser. No. 62/365,615, filed Jun. 30, 2016, and Non-provisional application Ser. No. 15/249,402, filed Aug. 27, 2016, herein incorporated in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC OR AS A TEXT FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB) Sequence Listing

The instant application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 21, 2016, is named PRODIF ST25.txt and is 112882 bytes in size.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINT INVENTOR

Not Applicable

BACKGROUND OF THE INVENTION (1) Field of the Invention

The presently disclosed subject matter generally relates to the treatment of disorders in a subject, including but not limited to pain. More particularly, the methods of the presently disclosed subject matter relate to the selection of agents for the treatment of pain, based on their ability to induce the differentiation of immature neuron cells, for the purpose of reducing pain and the susceptibility to pain.

(2) Description of Related Art

Not Applicable

TABLE OF ABREVIATIONS AAV adeno-associated virus ARTN artemin AV adenovirus BDNF brain-derived neurotrophic factor BSA bovine serum albumin CCI chronic constriction injury model of chronic pain, in which a ligature is tied to the sciatic nerve, inflicting chronic pain cDNA complementary DNA Cl chloride CNTF ciliary neurotrophic factor Contra Contralateral, on the opposite side DCX Doublecortin Dhh Desert hedgehog DNA deoxyribonucleic acid E. coli Escherichia coli EdU ethynyl-deoxyuridine EGF epidermal growth factor EPO erythropoietin FGF2 fibroblast growth factor 2 FZD frizzled receptor GABA gamma-aminobutiric acid GDL GDNF family of ligands GDNF glial cell derived neurotrophic factor GF growth factors HGF hepatocyte growth factor Hh Hedgehog Ihh Indian hedgehog INH inhibitors iPSCs induced pluripotent stem cells Ipsi ipsilateral, on the same side IUPAC International Union of Pure and Applied Chemistry K potassium ko gene knock-out LIF leukemia inhibitory factor Mash1 mammalian achaete-scute homolog 1 ml milliliter N3 Notch3 Na sodium NCC2 sodium-chloride cotransporter 2 NGF nerve growth factor NKCC1 sodium-potassium-chloride cotransporter 1 NRTN neurturin NT3 neurotrophin 3 NT4 neurotrophin 4 PBS phosphate buffer saline PSPN persephin PTCH Patched receptor Shh Sonic hedgehog SMO Smoothened receptor TetO tetracycline operator sequence TrkA tropomyosin receptor kinase A TrkB tropomyosin receptor kinase B TrkC tropomyosin receptor kinase C Veh saline vehicle solution Wnt Wingless WT wild-type

All amino-acid symbols used herein, including the Sequence Listing, are the three-letter abbreviations commonly used in the field of the invention.

BACKGROUND

Pain is an animal sensorial perception that indicates the presence of dangerous environmental conditions, prompting the organism to withdraw and adopt a protective stance in order to avoid injury, or to help heal an existing injury. The skin (epidermis and dermis) contains several types of receptors specialized in responses to mechanical, thermal and chemical (inflammation) stimuli, as well as poly-modal receptors. These receptors send nerve projections to the spinal cord, where the painful stimuli are processed, and a conscious or unconscious avoidance reaction is initiated.

The sensorial perception generated by specialized skin receptors and transmitted through nerves to the spinal cord is referred to as “nociception”. The processing of nociceptive information in the brain and its interaction with other cognitive and affective processes is referred to as “pain”. Since pain typically requires the input of nociceptive information, herein pain and nociception will be used interchangeably.

Pain can be classified as acute or chronic. Acute pain usually recedes after the elimination of the stimulus or healing of the injury. Chronic pain is often associated with chronic diseases, such as cancer or neurodegenerative disorders, but in many cases chronic pain can persist after the healing of the injury or disorder that have initiated it. Sometimes chronic pain can exist in the absence of any initiating conditions, such as fibromyalgia. Such cases are assumed to derive from genetic conditions. While acute pain is helpful in protecting the organism against injury, chronic pain has no benefit and interferes with normal activity. As a result, many classes of medication have been generated for the purpose of reducing or eliminating chronic pain.

All types of pain medication currently in use are targeted either against inflammation, or to reduce neuronal excitability by activating inhibitory receptors, such as GABA or opioid receptors, or by blocking the activation of excitatory sodium channels. Because of their mechanism of action, such medication is inevitably short-acting, effective only for as long as the active compound in the medication is bound to the receptor or ion channel, usually for several hours. Therefore, to maintain an analgesic effect, currently existing pain medication needs to be administered chronically throughout the duration of pain. Since such medication only addresses the symptoms of pain, not its cause, pain medication in general is considered to be “palliative”, not curative.

Chronic administration of a drug will inevitably lead to an increased expression of the receptor or ion channel which it targets, in order to compensate for the inhibition of its activity. This will require a continually increasing dose of medication in order to maintain the same level of analgesia, gradually leading to addiction. Addiction to analgesics is a frequent and serious health and social problem. Analgesics overdose can often lead to death.

Chronic pain caused by genetic factors often does not respond to any type of analgesic medication. In most cases, the genetic factors leading to chronic pain are unknown. Several transgenic mouse lines exist that show increased nociceptive sensitivity, including c-kit (Milenkovic, et al., Neuron 56: 893-906 (2007)), aldehyde dehydrogenase-2 (Zambelli, et al., Sci Transl Med. 6: 251ra118 (2014)), Notch3 ko (Rusanescu, et al., J Cell Mol Med. 18: 2103-16 (2014)) and Shp2 (Vegunta, et al., Am J Med Genet A. 167A: 2998-3005 (2015)).

Immature spinal cord neurons play a key role in the perception of pain (Rusanescu, et al., J Cell Mol Med. 19: 2352-2364 (2015)). Adult neurogenesis occurs in the spinal cord under normal conditions (Schechter, et al., Stem Cells. 25: 2277-2282 (2007); Horner, et al., J Neurosci. 20: 2218-28 (2000); Hugnot, et al., Frontiers Biosci. 16, 1044-59 (2011)), and is often amplified under pathological conditions such as injury (Rusanescu, et al., J Cell Mol Med. 19: 2352-2364 (2015)) or neurodegenerative disorders (Chi, et al., Stem Cells, 24: 34-43 (2006); Danilov, et al. Eur J Neurosci. 23, 394-400 (2006)). In one example, experimental chronic constriction injury of the sciatic nerve (CCI) induces significant cell proliferation in the spinal cord half ipsilateral to the injured nerve (FIG. 1). As a result, immature neurons are constantly generated and accumulate in the upper layers of the spinal cord dorsal horn (layers I-IV), responsible for nociceptive signaling (Rusanescu, et al., J Cell Mol Med. 19: 2352-2364 (2015)).

Immature neurons have high intracellular [Cl⁻] concentration because of an increased expression of the Na+K+2Cl-cotransporter NKCC1 (Yamada et al., J Physiol. 557: 829-41 (2004)), and therefore depolarize in response to GABA during their early development (LoTurco et al., Neuron 15: 1287-1298 (1995)). In addition, immature neurons have higher resting membrane potential (−50 mV) and higher excitability (Belleau et al., J Neurophysiol. 84: 2204-16 (2000); Ben-Ari et al., Physiol Rev. 87, 1215-84 (2007)). Upon maturation, changes in the relative expression of NKCC1/KCC2 cotransporters result in decreased intracellular Cl⁻ concentration and a hyperpolarizing response to GABA (Yamada et al., J Physiol. 557: 829-41 (2004)). A similar NKCC1/NCC2 expression reversal occurs in spinal cord dorsal horns after peripheral nerve injury (Price et al., Curr Top Med Chem. 5: 547-55 (2005); Lu et al, 2008), suggesting that these cells are immature neurons (Rusanescu, et al., J Cell Mol Med. 19: 2352-2364 (2015)).

Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4), have very similar neurotrophic and neuronal differentiation effects. Neurotrophins have been considered since their discovery to be promoters of nociception (Lewin et al., Trends Neurosci. 16: 353-359 (1993); Jankovski et al., Transl Pain Res. Chapter 2 (2010); Khan et al., Molecules. 20: 10657-88 (2015)). Although this increased nociception only occurs in the short term (hours to days), the pro-nociceptive role of neurotrophins is generally accepted as a scientific standard. Based on this idea, pain therapies currently under development are focusing on the inhibition of neurotrophin signaling (Shelton, J Peripher Nery Syst. 19 Suppl 2:S12-3 (2014)).

Ciliary neurotrophic factor (CNTF) has a similar neurotrophic and differentiating function to neurotrophins, and has been considered to have a similar role in pain physiology. Current attempts to treat pain have included the inhibition of CNTF signaling (Yang et al., Curr Gene Ther. 14:377-88 (2014)).

The glial-derived neurotrophic factor family of ligands (GDL), including glial cell-derived neurotrophic factor (GDNF), artemin (ARTN), neurturin (NRTN) and persephin (PSPN), have a similar neurotrophic and differentiating function to neurotrophins, and are considered to have a pain-inducing effect similar to neurotrophins. As a result, current strategies in pain treatment include the inhibition of GDL signaling (Merighi, Expert Opin Ther Targets 20:193-208 (2016); Lippoldt et al., Proc Natl Acad Sci USA 113:4506-11 (2016)).

The leukemia inhibitory factor (LIF) is considered to be a promoter of pain, in conjunction with, or independently of neurotrophins (Thompson et al., Neuroscience 71:1091-94 (1996); Engert et al., Neuropeptides 42:193-7 (2008)). At the same time, LIF is also a promoter of neuronal differentiation (Majumder et al., Stem Cells 30:2387-99 (2012)).

Angiotensin is known to be a promoter of pain (Marion et al., Cell 157:1565-76 (2014)), and at the same time it is an inducer of neuronal differentiation (Li et al., Mol Endocrinol. 21:499-511 (2007)).

Wnt is known to be a promoter of pain (Shi et al., Mol Pain 8:47 (2012); Liu et al., Pain 156:2572-84 (2015)), and at the same time it is an inducer of neuronal differentiation (Inestrosa et al., Cell Tissue Res. 359:215-23 (2015)).

Hh is known to be a promoter of pain (Babcock et al., Curr Biol. 21:1525-33 (2011)), and at the same time it is an inducer of neuronal differentiation (Dessaud et al., Development 135:2489-502 (2008)).

BRIEF SUMMARY OF THE INVENTION

The increase in nociceptive sensitivity after experimental spinal nerve injury (Bennett et al., Pain. 33: 87-107 (1988)) correlates with the increase in the number of spinal cord dorsal horn immature neurons (Rusanescu, et al., J Cell Mol Med. 19: 2352-2364 (2015)). This suggests that sensitivity to pain is determined by the number of highly excitable immature neurons in the dorsal horn. As a result, according to this model, any treatments that induce the differentiation of the hyper-excitable spinal cord immature neurons into mature neurons with low excitability would have an analgesic effect.

The idea that the level of pain is regulated by the number of immature spinal cord neurons is supported by the observation that all the proteins implicated in genetic models of pain, as described in paragraph [0009], are also coincidentally involved in neuronal differentiation: c-kit (Zhang, et al., Dev Neurosci. 31: 202-11 (2009)), aldehyde dehydrogenase-2 (Wallen A, et al., Exp Cell Res. 253, 737-46 (1999)), Notch3 (Rusanescu, et al., J Cell Mol Med. 18: 2103-16 (2014)), and Shp2 (Hadari Y R, et al., Mol Cell Biol. 18: 3966-73 (1998)). This idea was suggested by the correlation between the increased number of spinal cord immature neurons and increased pain sensitivity in Notch3 ko mice (Rusanescu, et al., J Cell Mol Med. 18: 2103-16 (2014)).

The present invention describes the use of agents that promote neuronal differentiation as a new class of analgesic drugs, the action of which is based on inducing the long-term differentiation of the highly excitable spinal cord immature neurons and thus reducing pain sensitivity. In the examples shown in this application, the said compounds reduce or eliminate pain in the long term. This effect is only apparently in contradiction with current knowledge that the same agents produce short-term pain.

The invention includes the use of polypeptides identical or similar to human neurotrophins, including BDNF (SEQ ID NO:1), NGF (SEQ ID NO:2), NT3 (SEQ ID NO:3), and NT4 (SEQ ID NO:4), in the treatment of pain. Another embodiment of this invention comprises the use of synthetic, semi-synthetic or natural molecules, which maintain functional similarity with human BDNF, NGF, NT3, or NT4, in activating members of the Trk family of receptors, for the purpose of treating pain.

Another embodiment of the invention includes the use of polypeptides identical or similar to the human CNTF (SEQ ID NO:5) in the treatment of pain. Another embodiment of this invention comprises the use of synthetic, semi-synthetic or natural molecules, which maintain functional similarity with human CNTF in activating the CNTF receptor, for the purpose of pain treatment.

Another embodiment of the invention includes the use of polypeptides identical or similar to the human GDNF family of ligands (GDL), including GDNF (SEQ ID NO:6), artemin (ARTN) (SEQ ID NO:7), neurturin (NRTN) (SEQ ID NO:8), and persephin (PSPN) (SEQ ID NO:9) in the treatment of pain. Another embodiment of this invention comprises the use of synthetic, semi-synthetic or natural molecules, which maintain functional similarity with human GDNF, ARTN, NRTN, or PSPN, in activating the RET receptor for the purpose of pain treatment.

Another embodiment of the present invention includes the use of a polypeptide identical or similar to the human leukemia inhibitory factor (LIF) (SEQ ID NO:10), in the treatment of pain. Another embodiment of this invention comprises the use of synthetic, semi-synthetic or natural molecules which maintain functional similarity with human LIF in activating the LIF receptor for the purpose of pain treatment.

Another embodiment of the present invention includes the use of a polypeptide identical or similar to the human angiotensin II (SEQ ID NO:11), in the treatment of pain. Another embodiment of this invention comprises the use of synthetic or natural molecules which maintain functional similarity with human angiotensin II in activating the angiotensin receptors, for the purpose of pain treatment.

Another embodiment of the present invention comprises the use of synthetic or natural molecules which promote directly or indirectly the activation of the c-Met receptor, for the purpose of pain treatment.

Another embodiment of the present invention includes the use of polypeptides identical or similar to the human Wnt, including Wnt1 (SEQ ID NO:12), Wnt2 (SEQ ID NO:13), Wnt2b (SEQ ID NO:14), Wnt3 (SEQ ID NO:15), Wnt4 (SEQ ID NO:16), Wnt5a (SEQ ID NO:17), Wnt5b (SEQ ID NO:18), Wnt6 (SEQ ID NO:19), Wnt7a (SEQ ID NO:20), Wnt7b (SEQ ID NO:21), Wnt8a (SEQ ID NO:22), Wnt8b (SEQ ID NO:23), and Wnt9a (SEQ ID NO:24), in the treatment of pain. Another embodiment of this invention comprises the use of synthetic, semi-synthetic or natural molecules which have functional similarity with human Wnt in promoting the activation of the frizzled (FZD) receptors for the purpose of pain treatment.

Another embodiment of the present invention includes the use of polypeptides identical or similar to the human Hh, including Shh (SEQ ID NO:25), Dhh (SEQ ID NO:26), and Ihh (SEQ ID NO:27) in the treatment of pain. Another embodiment of this invention comprises the use of synthetic or natural molecules which maintain functional similarity with human Hh in promoting the activation of the Smoothened (SMO) receptor for the purpose of pain treatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the quantification of immunofluorescence staining of cell proliferation marker EdU in rat spinal cord slices, before (week 0) and at 2, 4 weeks after unilateral CCI. EdU staining was quantified as a percentage of the grey matter area, in the ipsilateral and contralateral halves of rat spinal cord. EdU staining is substantially increased ipsilaterally four weeks after CCI, at a time when inflammation-induced gliosis is reduced back to background level. This suggests that at least part of the EdU-stained proliferating cells reflect adult neurogenesis.

FIG. 2 depicts the quantification of nestin expression in rat spinal cord dorsal horns, before (week 0) and at 2, 4, 6 weeks after unilateral CCI. Nestin expression was quantified by immunofluorescence, as a percentage of ipsilateral and contralateral dorsal horn areas, respectively. Nestin is a marker for neuron precursor cells.

FIG. 3 depicts the quantification of Mash1 expression in rat spinal cord gray matter, before (week 0) and at 2, 4, 6 weeks after unilateral CCI. Mash1 expression was quantified by immunofluorescence, as a percentage of the ipsilateral and contralateral halves of the spinal cord gray matter area. Mash1 expression peaks at week 4 after CCI, which coincides with the time required for the newly generated neural progenitor cells to migrate from the central canal of the spinal cord to the upper dorsal horn. The contralateral increase in Mash1 expression is localized for the most part in the ventral horn and therefore is irrelevant for pain perception. Mash1 is a marker for neuron precursor cells.

FIG. 4 depicts the quantification of doublecortin (DCX) expression in rat spinal cord gray matter, before (week 0) and at 2, 4, 6 weeks after unilateral CCI. Doublecortin expression was quantified by immunofluorescence, as a percentage of the ipsilateral and contralateral halves of the spinal cord gray matter area. Doublecortin is a marker for immature neurons.

FIG. 5 depicts the quantification of Notch3 expression in rat spinal cord gray matter, before (week 0) and at 2, 4, 6 weeks after unilateral CCI. Notch3 expression was quantified by immunofluorescence, as a percentage of the ipsilateral and contralateral halves of the spinal cord gray matter area. Notch3 is a marker for neuron progenitor cells and immature neurons.

FIG. 6 depicts the increase in the number of NeuN-positive mature neurons in the ipsilateral spinal cord dorsal horn layers I and II, six weeks after unilateral CCI. NeuN expression, which is a marker for mature neurons, was quantified by immunofluorescence.

FIG. 7 depicts the weekly variation of nociceptive sensitivity on the ipsilateral (Ipsi, A and B) and contralateral hind paws (Contra, C and D) of rats subjected to unilateral CCI. Mechanical nociceptive sensitivity shown in A and C was measured using a von Frey series of filaments calibrated in grams. Thermal nociceptive sensitivity shown in B and D was measured in seconds as the withdrawal time after a beam of radiant light was applied to the paw. Arrows shown in A, B and C indicate inflexion points in the graph, which suggest a change in mechanism. The CCI graphs are depicted in comparison to reference graphs (Sham), which represent nociceptive sensitivity in rats subjected to sham surgery (surgery was performed as in CCI rats, but without performing nerve constriction). Asterisks indicate the statistical probability that the inflection points are a random occurrence. Using a generally accepted convention, “*” indicates a statistical probability 0.01<P<0.05, “**” indicates a statistical probability 0.001<P<0.01 and “***” indicates a statistical probability P<0.001. Error bars indicate standard error.

FIG. 8 depicts the correlation between the number of immature neurons present in the spinal cord and the changes in nociceptive sensitivity. (A) depicts the variation in the expression of immature neuron markers Mash1, doublecortin and Notch3 in rat spinal cord ipsilateral and contralateral gray matter halves, 4 weeks after the rats were subjected to CCI and treated either with spinal (intrathecal) injections of saline (CCI+Veh), of inhibitors for EGF and FGF2 (CCI+INH), or of recombinant EGF and FGF2 (CCI+GF). Marker expression was quantified by immunofluorescence, as a percentage of the areas of the ipsilateral and contralateral gray matter halves, respectively. (B) depicts the weekly variation of the mechanical nociceptive sensitivity, measured in grams of pressure, on the paw ipsilateral to CCI, after injection with Veh or INH. (C) depicts the weekly variation of the thermal nociceptive sensitivity, measured in seconds of withdrawal latency, on the paw ipsilateral to CCI, after injection with Veh or INH. (D) depicts the weekly variation of the mechanical nociceptive sensitivity, measured in grams of pressure, on the paw ipsilateral to CCI, after injection with Veh or GF. (E) depicts the weekly variation of the thermal nociceptive sensitivity, measured in seconds of withdrawal latency, on the paw ipsilateral to CCI, after injection with Veh or GF. Statistical analysis is depicted as in FIG. 7.

FIG. 9 depicts the effect of BDNF treatment on the number of spinal cord immature neurons in parallel with its effect on nociceptive sensitivity. (A) depicts the variations in the expression of immature neuron markers Mash1, doublecortin and Notch3 in rat spinal cord ipsilateral and contralateral gray matter halves, 6 weeks after the rats were subjected to CCI and treated with intrathecal injections of saline (CCI+Veh) or BDNF (CCI+BDNF). Marker expression is quantified by immunofluorescence, as a percentage of the areas of the ipsilateral and contralateral gray matter halves, respectively. (B) depicts the weekly variation of the mechanical nociceptive sensitivity on the paw ipsilateral to CCI after injection with Veh or BDNF, measured in grams of pressure. (C) depicts the weekly variation of the thermal nociceptive sensitivity on the paw ipsilateral to CCI, measured in seconds of withdrawal latency, after injection with Veh or BDNF. (D) depicts the weekly variation of the mechanical nociceptive sensitivity on the paw ipsilateral to CCI, measured in grams of pressure, when the BDNF treatment was delayed for 3 weeks after CCI. (E) depicts the weekly variation of the thermal nociceptive sensitivity on the paw ipsilateral to CCI, measured as seconds of withdrawal latency, when the BDNF treatment was delayed for 3 weeks after CCI.

FIG. 10 depicts the effect of 7,8-dixydroxyflavone (DHF) treatment on the number of spinal cord immature neurons in parallel with its effect on nociceptive sensitivity. (A) depicts the variations in the expression of immature neuron markers Mash1, doublecortin and Notch3 in rat spinal cord ipsilateral and contralateral gray matter halves, 6 weeks after the rats were subjected to CCI and treated with intraperitoneal injections of saline (CCI+Veh) or 7,8-dihydroxyflavone (CCI+DHF). Marker expression is quantified by immunofluorescence, as a percentage of the areas of the ipsilateral and contralateral gray matter halves, respectively. (B) depicts the weekly variation of the mechanical nociceptive sensitivity on the paw ipsilateral to CCI after injection with Veh or DHF, measured in grams of pressure. (C) depicts the weekly variation of the thermal nociceptive sensitivity on the paw ipsilateral to CCI after injection with Veh or DHF, measured in seconds of withdrawal latency. (D) depicts the weekly variation of the mechanical nociceptive sensitivity on the paw ipsilateral to CCI, measured in grams of pressure, when the DHF treatment was delayed for 3 weeks after CCI. (E) depicts the weekly variation of the thermal nociceptive sensitivity on the paw ipsilateral to CCI, measured as seconds of withdrawal latency, when the DHF treatment was delayed for 3 weeks after CCI.

DETAILED DESCRIPTION

In subjects with injuries, disabilities, disorders or diseases, including but not limited to neurological, sensory disorders, psychiatric disorders, diabetes, rheumatism, cancer, and other diseases, alterations in cellular numbers and/or activity can occur. Some of these alterations may involve an increase in the number of adult neural stem cells. During the differentiation process, before becoming mature neurons, the neural stem cells transit a stage of immature neurons, characterized by increased excitability. This invention is based on the idea that, when immature neurons integrate into neuron networks responsible for pain perception and processing, their increased excitability is perceived as pain. Accordingly, by providing subjects suffering from such disorders with a method of treatment that accelerates the neuronal differentiation process and reduces the number of hyper-excitable immature neurons, the symptoms of pain can be alleviated or eliminated. As disclosed for the first time herein, many classes of promoters of neuronal differentiation have a long-term analgesic effect, by reducing the number of immature neurons present in the neural circuits responsible for pain perception and processing.

Another embodiment of the present invention includes the use of promoters of neuronal differentiation in the treatment of pain resulting from incomplete or defective cellular differentiation due to genetic variations. In such patients, a large number of hyper-excitable immature neurons is always present even in the absence of any noticeable injury or disease, contributing to a chronically increased pain sensitivity. As a result of this increased pain sensitivity, the affected individual perceives as painful stimuli which are not normally perceived as painful in a normal individual. Promoters of neuronal differentiation can alleviate or eliminate pain in such cases, for which no alternative treatment exists.

All publications mentioned herein are incorporated by reference to the extent allowed by the law for the purpose of describing and disclosing the proteins, peptides, chemical molecules, vectors, cells and methodologies reported therein that might be used with the present invention. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Definitions

This invention is not limited to the methods, protocols, molecules, cell lines, vectors or reagents described herein because they may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the reach of the present invention. Although any materials and methods similar or equivalent to those described herein can be used in the practice of the present invention, representative materials and methods are described herein.

Following patent law convention, the terms “a”, “an”, and “the” refer to “one or more”, e.g. reference to “a compound” includes a plurality of compounds. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention.

Unless otherwise indicated, all numbers expressing quantities of ingredients, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, the numerical parameters set forth in the current specification are approximations that can vary depending on the desired properties sought by the presently disclosed subject matter. Furthermore, Applicants desire that the following terms be given the particular definition as defined below.

The term “agent” shall be construed to include proteins, polypeptides, peptides, chemical molecules and compounds that are capable of promoting or inducing neuronal differentiation, for the purpose of pain treatment.

The term “sequence” shall be construed to include any natural or synthetic amino-acid or nucleotide sequence that maintains, fully or partially, functional similarity with the human molecules indicated herein. Because of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding these proteins, or fragments thereof, may be used. Nucleotide sequences may vary by selecting nucleotide combinations based on possible codon choices, in accordance with standard triplet genetic codes.

The term “sequence homology” shall be construed as meaning the percentage of the amino-acid residues in the candidate sequence that are similar with the residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps if necessary to achieve the maximum percentage identity for the entire sequence. Amino-acid similarity shall be determined according to one of several published physico-chemical criteria, selected from the group comprising hydrophobicity, aromaticity, basicity, acidity, and polarity, well known to those of ordinary skill in the art in the field of the invention.

The term “functional similarity” with respect to the amino-acid sequence of a ligand shall be construed to include any natural or synthetic molecule that maintains, fully or partially, the ability to bind to, to activate, or both, specific cellular receptors in a manner similar to its natural ligand, and leading to a similar cellular outcome, including but not limited to neuronal differentiation. Because of the similarity in physicochemical properties between amino-acids, several amino-acids may be used interchangeably for each position in the sequence of a protein to generate alternate sequences that maintain similar function to the natural protein, as commonly understood by one of ordinary skill in the art in the field of the invention. The term shall not be construed to be limited to specific percentage ranges of sequence identity or similarity with the proteins indicated herein, as the introduction of gaps, insertions, substitutions or extensions in an amino-acid or nucleotide sequence may substantially change the percentage of sequence identity or homology, but may maintain the same functionality as the original protein sequence.

The term “neuronal differentiation” shall be construed to represent the process of converting stem cells, neuronal stem cells, immature neurons, or any other type of incompletely differentiated neuron progenitors, into fully functional and physiologically integrated neurons, as commonly understood by one of ordinary skill in the art in the field of the invention. A completely or fully differentiated neuron is a cell that has reached a state characterized by a maximal, final functional role in comparison to the other cells with a similar phenotype present in an organism. For example, a fully differentiated neuron is a cell that expresses a typical set of genes, has a typical electrophysiological response and performs a typical physiological function, usually as part of a cellular network, as understood by those of ordinary skill in the art. The neuronal differentiation activity of a candidate agent (polypeptide, molecule or chemical compound) can be measured in cell culture, in an organism or tissue, by comparing the neuronal differentiation level between cells treated with the candidate agent, cells treated with a known agent inducer of neuronal differentiation (for example BDNF), and cells treated with inactive molecules used as control or reference. The neuronal differentiation level can be determined through any method commonly known to one of ordinary skill in the art in the field of the invention, including but not limited to the measurement of neurite length, electrophysiological recording, immunofluorescence staining of specific markers, cell sorting, gene expression, spectroscopic measurement of bound ligands, radioactive labeling, cell secretion, imaging, organism behavior, etc.

A cell may present various degrees or levels of differentiation, which is a continuous, not a punctual process. For example, progenitor cells display some degree of differentiation relative to stem cells, as progenitor cells can produce by differentiation only a subset of the cells that stem cells can produce. However, progenitor cells maintain the ability to divide into identical daughter cells, similar to stem cells. Immature cells are construed as including cells that are characterized by an incomplete degree of differentiation. In addition, immature cells may display some phenotypical and physiological characteristics similar to the characteristics of un-differentiated cells, some characteristics similar to the characteristics of fully differentiated cells, as well as some unique characteristics that are different from both un-differentiated and fully differentiated cells. For example, immature neurons are neurons that express a subset of the genes typically expressed in neural progenitor cells, as well as some of the genes expressed in fully differentiated neurons, and a set of genes that are expressed neither in neural progenitor cells, nor in fully differentiated neurons. In addition, immature neurons display unique electrophysiological properties, including increased excitability, as commonly known to those of ordinary skill in the art. For example immature neurons produce an electric response when stimulated with the amino acid gamma-aminobutiric acid (GABA), which reflects the presence of GABA receptors and ion channel proteins similar to neurons, however this electric response produced has the opposite sign relative to the electric response produced by fully differentiated or mature neurons.

The term “ligand” as used herein shall be construed to include natural or artificial molecules, including but not limited to proteins, polypeptides, peptides, animal or plant-made molecules or semi-synthetic molecules, which bind, directly or indirectly, to a specific human cellular receptor, selected from the group consisting of: TrkA, TrkB, TrkC, CNTF receptor, GDL family of receptors, Ret receptor, LIF receptor, c-Met receptor, angiotensin receptor, erythropoietin receptor, frizzled (FZD), patched (PTCH), TNF-alpha receptor, and other receptors, and initiate or promote a physiological process, including but not limited to neuronal differentiation. For example, the term “ligand” will also include molecules that bind to a receptor indirectly, by mediation of another molecule or molecules, and promote the physiological function of that receptor through a physico-chemical process selected from a group which includes promoting ligand-receptor binding, post-translational modification of the ligand or receptor, and allosteric modulation of ligand-receptor binding. The term “ligand” will also include antibodies generated against a specific human receptor described herein, and which induce receptor activation. Furthermore, the term “ligand” shall include poly-peptides that have 90%-100%, 80%-90%, or 70%-80% homology to a poly-peptide selected from the group consisting of: NGF, BDNF, NT3, NT4, CNTF, GDNF, ARTN, NRTN, PSPN, LIF, angiotensin, erythropoietin, HGF, Wnt, and Hh.

The term “neurotrophin” shall be construed as a protein selected from a group that includes: NGF, BDNF, NT3, and NT4. Neurotrophins bind to, and activate the Trk family of receptor tyrosine kinases, which includes TrkA, TrkB, TrkC and the low-affinity receptor p75trk. Trk receptors activate multiple down-stream signaling pathways, including but not limited to: Ras, PI3-kinase and Ral. Each said pathway can be modulated independently, leading to a variety of biological effects, selected from a group which includes neuronal survival, neuronal differentiation, oncogenesis, neuronal apoptosis, gene regulation, neuronal communication, and many others. Due to the multiple signaling pathways induced by neurotrophins through the activation of Trk receptors, said biological effects can be regulated independently of each other, in conjunction with other additional cellular signals, although some of these biological effects may occur concurrently. For example, neurotrophins may induce at the same time neuronal differentiation and neuronal survival, however these two processes are regulated by different combinations of Trk-dependent pathways, and in conjunction with other, distinct Trk-independent pathways. As a consequence of such differences, neuronal differentiation and neuronal survival are distinct biological processes, and their potential correlation in time does not imply an interdependence with one another.

The terms “promote” and “promotion” used in reference to neuronal differentiation shall be construed as a cellular action selected from the group consisting of: initiation, acceleration, contribution, assistance, induction, and stimulation, of neuronal differentiation.

The terms “promoter” and “inducer” used in reference to neuronal differentiation shall be construed as an agent selected from the group consisting of: a polypeptide, a molecule and a chemical compound, having the property of promoting, inducing, initiating, or accelerating neuronal differentiation. This action can be performed by activating a receptor, a signaling pathway or both, including but not limited to TrkA, TrkB, TrkC, a CNTF receptor, a GDL receptor, a LIF receptor, an angiotensin receptor, an EPO receptor, a FZD receptor, and a SMO receptor.

The term “BDNF” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human BDNF, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “BDNF” or not. Functional similarity to BDNF comprises the ability of an agent to bind to, and activate the TrkB receptor, and to promote neuronal differentiation. While a key receptor binding site is located between amino-acids 173-178 of human BDNF (Obianyo et al., Biochim Biophys Acta 1834:2213-2218 (2013)), other domains of BDNF also contribute to ligand specificity and activity, and will therefore contribute to the activation of the TrkB receptor. Therefore BDNF fragments that retain only some of the functional domains present in full-length BDNF may still activate the TrkB receptor and induce neuronal differentiation. Several natural forms of BDNF exist, generated within the organism by cleavage from the endogenously synthesized full length BDNF precursor. For example, the human precursor BDNF (32 kDa) is a polypeptide 247 amino-acids long. Enzymatic cleavage of precursor BDNF between amino-acids 57-58 generates one form of BDNF (28 kDa) (Mowla et al., J Biol Chem. 276:12660-12666 (2001)). A different cleavage of precursor BDNF between amino-acids 128-129 generates “mature” BDNF (14 kDa) (Seidah et al., J Biol Chem. 379:247-50 (1996)). For example, a polypeptide depicted in the amino-acid sequence “BDNF FD” (SEQ ID NO:1), located between amino-acids 129-247 of human BDNF precursor, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 96%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Gallus gallus and Xenopus laevis. Many other amino-acid sequences derived from BDNF FD (SEQ ID NO:1) may be generated, which maintain functional similarity with BDNF and may be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from BDNF FD, may generate polypeptides with functional similarity to BDNF, which may be used in the treatment of pain. In another example, a chemically modified peptide comprising only two amino-acid residues can maintain the ability to activate the TrkB receptor (Obianyo et al., Biochim Biophys Acta 1834:2213-2218 (2013)). These examples indicate that peptides of any length that maintain 90%-100% homology with BDNF FD may maintain the ability to activate the TrkB receptor even in the absence of some of the functional domains present in BDNF, and therefore may be used in the treatment of pain. For example the polypeptide BDNF FD1 (SEQ ID NO:28), obtained by removing 2 amino-acids from BDNF FD, maintains functional similarity with BDNF and may be used for the activation of TrkB and for the treatment of pain. Furthermore, the term “BDNF” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of BDNF FD or BDNF FD1. For example a polypeptide having the BDNF FD sequence can be synthesized using synthetic chemistry or can be generated by a cell in-vitro, or it can be produced within an organism from the equivalent oligonucleotide sequence (cDNA).

The term “NGF” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human NGF, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “NGF” or not. Functional similarity to NGF comprises the ability of an agent to bind to, and activate, fully or partially, the TrkA receptor, and to promote neuronal differentiation. Several natural forms of NGF exist, generated within the organism by cleavage from the endogenously synthesized full length NGF precursor. For example, the human precursor of NGF (pre-proNGF) is a polypeptide 241 amino-acids long. Enzymatic cleavage of precursor NGF between amino-acids 121-122 generates “mature” NGF (beta-NGF, 13.5 kDa) (Seidah et al., Biochem J. 314, 951-960 (1996)). For example, a polypeptide comprising the amino-acid sequence “NGF FD” (SEQ ID NO:2), located between amino-acids 122-241 of human NGF precursor, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 92%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Gallus gallus and Lipotes vexillifer. Many other amino-acid sequences derived from NGF FD may be generated, which maintain functional similarity with NGF and may be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from NGF FD, may generate polypeptides with functional similarity to NGF, which may be used in the treatment of pain. These examples indicate that polypeptides of any length that maintain at least 90% homology with NGF FD, may also maintain the ability to activate the TrkA receptor, and may be used in the treatment of pain. For example the polypeptide NGF FD1 (SEQ ID NO:29), obtained by removing 2 N-terminal amino-acids from NGF FD, maintains all the functional domains present in NGF FD and may be used for the activation of TrkA and for the treatment of pain. Furthermore, the term “NGF” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of NGF FD or NGF FD1.

The term “NT3” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human neurotrophin-3 (NT3), as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “NT3” or not. Functional similarity to NT3 comprises the ability of an agent to bind to, and activate, fully or partially, the TrkC or TrkB receptors, and to promote neuronal differentiation. The endogenously synthesized human NT3 (pre-NT3) comprises two precursor protein isoforms 270 and 257 amino-acids long, respectively. Enzymatic cleavage of pre-NT3 (between amino-acids 138-139 in the case of isoform 2) generates “mature” NT3 (Seidah et al., FEBS Lett. 379:247-250 (1996)). For example, a polypeptide comprising the amino-acid sequence “NT3 FD” (SEQ ID NO:3), located between amino-acids 139-257 of human NT3 precursor isoform 2, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 99%-100% identity among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Gallus gallus and Xenopus tropicalis. Many other polypeptides derived from NT3 FD may be generated, which maintain functional similarity with NT3 and may be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from NT3 FD, may generate agents with functional similarity to NT3, which may be used in the treatment of pain. These examples indicate that polypeptides of any length that maintain at least 90% homology with SEQ ID NO:3, may also maintain the ability to activate the TrkC or TrkB receptor, and may be used in the treatment of pain. For example the polypeptide NT3 FD1 (SEQ ID NO:30), obtained by removing 2 N-terminal amino-acid residues from NT3 FD, maintains all the functional domains present in NT3 FD and may be used for the activation of TrkC or TrkB, and for the treatment of pain. Furthermore, the term “NT3” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of NT3 FD or NT3 FD1.

The term “NT4” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human neurotrophin-4 (NT4), as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “NT4” or not. Functional similarity to NT4 comprises the ability of an agent to bind to, and to activate, fully or partially, the TrkB receptor, and to promote neuronal differentiation for the purpose of treating pain. The endogenously synthesized human NT4 (pre-NT4) comprises two precursor protein isoforms X1, 220 amino-acids long, and X2, 210 amino-acids long. Enzymatic cleavage of pre-NT4 (between amino-acids 80-81 in the case of isoform X2) generates “mature” NT4, which is considered the biologically active form of NT4. For example, a polypeptide comprising the amino-acid sequence “NT4 FD” (SEQ ID NO:4), located between amino-acids 81-210 of human NT4, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 98%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, and Sus scrofa. Many other polypeptides derived from NT4 FD can be generated, which may maintain functional similarity with NT4 and may be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from NT4 FD, may generate polypeptides with functional similarity to NT4, which may be used in the treatment of pain. These examples suggest that polypeptides of any length that maintain at least 90% homology with SEQ ID NO:4, may also maintain the ability to activate the TrkB receptor, and may be used in the treatment of pain. For example the polypeptide NT4 FD1 (SEQ ID NO:31), obtained by removing 2 N-terminal amino-acids from NT4 FD, maintains all the functional domains present in NT4 FD and may be used for the activation of TrkB, and for the treatment of pain. Furthermore, the term “NT4” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequences of NT4 FD or NT4 FD1.

The term “CNTF” shall be construed as including natural and artificial amino-acid sequences that maintain sequence or functional similarity, or both, to human CNTF, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “CNTF” or not. Functional similarity to CNTF comprises the ability of an agent to bind to, and activate, fully or partially, the CNTF receptor, and to promote neuronal differentiation. For example, a polypeptide comprising the amino-acid sequence “CNTF FD” (SEQ ID NO:5), located between amino-acids 1-200 of human CNTF, may be used to promote neuronal differentiation for the purpose of treating pain. This sequence is highly conserved, having 93%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, and Sus scrofa. Many other polypeptides derived from CNTF FD can be generated, which may maintain functional similarity with CNTF, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from CNTF FD, may generate polypeptides with functional similarity to CNTF, which may be used in the treatment of pain. These examples suggest that polypeptides of any length that maintain at least 90% homology with SEQ ID NO:5, may also maintain the ability to activate the CNTF receptor, and may be used in the treatment of pain. For example the polypeptide CNTF FD1 (SEQ ID NO:32), obtained by removing 2 amino-acid residues from CNTF FD, maintains the functional similarity with CNTF and may be used for the activation of the CNTF receptor, and for the treatment of pain. In another example, cintrophin, a polypeptide comprising amino-acid residues 148-161 of human CNTF, maintains functional similarity with CNTF (turn et al., J Neurosci Res. 44:133-41 (1996)). Furthermore, the term “CNTF” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequences of CNTF FD or CNTF FD1.

The term “GDNF” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human GDNF, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “GDNF” or not. Functional similarity to GDNF comprises the ability of an agent to bind to, and activate the GDNF family receptor alpha-1 and the Ret receptor, and to promote neuronal differentiation. Human GDNF comprises at least 5 isoforms, ranging from 159 to 228 amino-acids. For example, a polypeptide comprising the amino-acid sequence “GDNF FD” (SEQ ID NO:6), located between amino-acids 78-211 of human GDNF precursor isoform 1, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 95%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, and Canis lupus. Many other polypeptides derived from GDNF FD can be generated, which may maintain functional similarity with GDNF, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from GDNF FD, may generate polypeptides with functional similarity to GDNF, which may be used in the treatment of pain. These examples suggest that polypeptides of any length that maintain at least 90% homology with GDNF FD, may also maintain the ability to activate the GDNF family receptor alpha-1, and may therefore be used in the treatment of pain. For example the polypeptide GDNF FD1 (SEQ ID NO:33), obtained by removing 2 amino-acid residues from GDNF FD, maintains functional similarity GDNF and may be used for the activation of the GDNF family receptor alpha-1, and for the treatment of pain. In another example, gliafin, a polypeptide comprising amino-acid residues 153-167 of GDNF, maintains functional similarity with GDNF (turn et al., J Neurosci Res. 44:133-41 (1996)). Furthermore, the term “GDNF” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of GDNF FD.

The term “ARTN” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human ARTN, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “ARTN” or not. Functional similarity to ARTN shall be construed as the ability of an agent to bind to, and activate the GDNF family receptor alpha-3 and the Ret receptor, and to promote neuronal differentiation. Human ARTN comprises at least 3 isoforms. For example, a polypeptide comprising the amino-acid sequence “ARTN FD” (SEQ ID NO:7), located between amino-acids 108-220 of human ARTN precursor isoform 1, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 95%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, and Sus scrofa. Many other polypeptides derived from ARTN FD can be generated, which may maintain functional similarity with ARTN, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from ARTN FD, may generate polypeptides with functional similarity to ARTN, which may be used in the treatment of pain. These examples suggest that peptides of any length that maintain at least 90% homology with ARTN FD, may also maintain the ability to activate the GDNF family receptor alpha-3, and may therefore be used in the treatment of pain. For example the polypeptide ARTN FD1 (SEQ ID NO:34), obtained by removing 2 amino-acid residues from ARTN FD, maintains functional similarity with ARTN and may be used for the activation of the GDNF family receptor alpha-3, and for the treatment of pain. Furthermore, the term “ARTN” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of ARTN FD.

The term “NRTN” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human NRTN, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “NRTN” or not. Functional similarity to NRTN shall be construed as the ability of an agent to bind to, and activate the GDNF family receptor alpha-2 and the Ret receptor, and to induce neuronal differentiation. For example, a polypeptide comprising the amino-acid sequence NRTN FD (SEQ ID NO:8), located between amino-acids 96-197 of human NRTN, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 98%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Canis lupus, and Tursiops truncatus. Many other polypeptides derived from NRTN FD can be generated, which may maintain functional similarity with NRTN, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from NRTN FD, may generate polypeptides with functional similarity to NRTN, which may be used in the treatment of pain. These examples suggest that peptides of any length that maintain at least 90% homology with NRTN FD, may also maintain the ability to activate the GDNF family receptor alpha-2, and may therefore be used in the treatment of pain. For example the polypeptide NRTN FD1 (SEQ ID NO:35), obtained by removing 2 amino-acid residues from NRTN FD, maintains functional similarity with NRTN and may be used for the activation of the GDNF family receptor alpha-2, and for the treatment of pain. Furthermore, the term “NRTN” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of NRTN FD.

The term “PSPN” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human PSPN, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “PSPN” or not. Functional similarity to PSPN shall be construed as the ability of an agent to bind to, and to activate the GDNF family receptor alpha-4 and the Ret receptor, and to induce neuronal differentiation. For example, a polypeptide comprising the amino-acid sequence “PSPN FD” (SEQ ID NO:9), located between amino-acids 22-156 of human PSPN, may be used to promote neuronal differentiation for the purpose of pain treatment. This sequence is highly conserved, having 92%-100% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Canis lupus, and Sus scrofa. Many other amino-acid sequences derived from PSPN FD can be generated, which may maintain functional similarity with PSPN, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from PSPN FD, may generate polypeptides with functional similarity to PSPN, which may be used in the treatment of pain. These examples suggest that peptides of any length that maintain at least 90% homology with PSPN FD, may also maintain the ability to activate the GDNF family receptor alpha-4, and may therefore be used in the treatment of pain. For example amino-acid sequence PSPN FD1 (SEQ ID NO:36), obtained by removing 2 amino-acid residues from PSPN FD, maintains functional similarity with PSPN and may be used for the activation of the GDNF family receptor alpha-4, and for the treatment of pain. Furthermore, the term “PSPN” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of PSPN FD.

The term “LIF” shall be construed as including natural and artificial polypeptides that maintain sequence or functional similarity, or both, to human LIF functional domains, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “LIF” or not. Functional similarity to LIF comprises the ability of an agent to bind to, and activate the LIF receptor, and to promote neuronal differentiation. Human LIF comprises at least 2 isoforms. For example, a LIF sequence comprising the amino-acid sequence LIF FD (SEQ ID NO:10), located between amino-acids 23-202 of human LIF isoform 1, may be used to promote neuronal differentiation for the purpose of treating pain. This sequence is highly conserved, having at least 93% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, and Canis lupus. Many other amino-acid sequences derived from LIF FD can be generated, which may maintain functional similarity with LIF, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from LIF FD, may generate amino-acid sequences with functional similarity to LIF, which may be used in the treatment of pain. These examples suggest that polypeptides of any length that maintain at least 90% homology with LIF FD, may also maintain the ability to activate the LIF receptor, and may therefore be used in the treatment of pain. For example amino-acid sequence LIF FD1 (SEQ ID NO:37), obtained by removing 2 amino-acid residues from LIF FD, maintains functional similarity with LIF and may be used for the activation of the LIF receptor, and for the treatment of pain. Furthermore, the term “LIF” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of LIF FD.

The term “angiotensin” shall be construed as including natural and artificial peptides that maintain sequence similarity to human angiotensin II, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “angiotensin II” or not. Functional similarity to angiotensin II shall be determined by the ability of an agent to bind to, and activate the angiotensin receptors, and to promote neuronal differentiation. For example angiotensin III and angiotensin IV retain some of the ability of angiotensin II to activate at least some of the angiotensin receptors. In one embodiment, an angiotensin sequence consisting of the amino-acid sequence “ANG” (SEQ ID NO:11), located between amino-acids 34-41 of human angiotensinogen, may be used to promote neuronal differentiation for the purpose of treating pain. This sequence is highly conserved, having at least 95% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, Gallus gallus and Canis lupus. In another embodiment, other amino-acid sequences derived from ANG can be generated, which may maintain functional similarity with angiotensin, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, or 6 amino-acid residues from ANG, may generate amino-acid sequences with functional similarity to angiotensin, which may be used in the treatment of pain. For example, angiotensin IV (ANG1, SEQ ID NO:38) retains functional similarity with angiotensin, and may be used in the treatment of pain. These examples suggest that polypeptides of any length that maintain at least 80% homology with ANG, may also maintain the ability to activate the angiotensin receptors, and may therefore be used in the treatment of pain. Furthermore, the term “angiotensin” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of angiotensin.

The term “Wnt” shall be construed as including natural and artificial polypepties that maintain sequence or functional similarity, or both, to any member of human Wnt family of ligands, comprising Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b and Wnt9a, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “Wnt” or not. Functional similarity to Wnt shall be construed as the property of an agent to bind to, and activate a FZD receptor, and to promote neuronal differentiation. In one embodiment, a polypeptide selected from the group comprising the amino-acid sequences WNT1 FD (SEQ ID NO:12) comprising amino-acids 28-370 of human Wnt1, WNT2 FD (SEQ ID NO:13) comprising amino-acids 26-360 of human Wnt2, Wnt2b FD (SEQ ID NO:14) comprising amino-acids 40-391 of human Wnt2b isoform 2, Wnt3 FD (SEQ ID NO:15) comprising amino-acids 22-355 of human Wnt3, Wnt4 FD (SEQ ID NO:16) comprising amino-acids 23-351 of human Wnt4, Wnt5a FD (SEQ ID NO:17) comprising amino-acids 62-380 of human Wnt5a, Wnt5b FD (SEQ ID NO:18) comprising amino-acids 18-359 of human Wnt5b, Wnt6 FD (SEQ ID NO:19) comprising amino-acids 25-365 of human Wnt6, Wnt7a FD (SEQ ID NO:20) comprising amino-acids 32-349 of human Wnt7a, Wnt7b FD (SEQ ID NO:21) comprising amino-acids 25-349 of human Wnt7b, Wnt8a FD (SEQ ID NO:22) comprising amino-acids 25-351 of human Wnt8a isoform 3, Wnt8b FD (SEQ ID NO:23) comprising amino-acids 23-351 of human Wnt8b, and Wnt9a FD (SEQ ID NO:24) comprising amino-acids 30-365 of human Wnt9a, may be used to promote neuronal differentiation for the purpose of treating pain. Sequences Wnt1 FD, Wnt2 FD, Wnt2b FD, Wnt3 FD, Wnt4 FD, Wnt5a FD, Wnt5b FD, Wnt6 FD, Wnt7a FD, Wnt7b FD, Wnt8a FD, Wnt8b FD, and Wnt9a FD are highly conserved, having at least 93% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, and Gallus gallus. In a different embodiment, many other polypeptides derived from a sequence selected from the group consisting of Wnt1 FD, Wnt2 FD, Wnt2b FD, Wnt3 FD, Wnt4 FD, Wnt5a FD, Wnt5b FD, Wnt6 FD, Wnt7a FD, Wnt7b FD, Wnt8a FD, Wnt8b FD and Wnt9a FD, can be generated that maintain functional similarity with Wnt. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from an amino-acid sequence selected from the group consisting of: Wnt1 FD, Wnt2 FD, Wnt2b FD, Wnt3 FD, Wnt4 FD, Wnt5a FD, Wnt5b FD, Wnt6 FD, Wnt7a FD, Wnt7b FD, Wnt8a FD, Wnt8b FD or Wnt9a FD, may generate polypeptides with functional similarity to Wnt, which may be used in the treatment of pain. These examples suggest that polypeptides of any length that maintain at least 90% homology with Wnt, may also maintain the ability to activate the frizzled receptors, and may therefore be used in the treatment of pain. For example amino-acid sequences Wnt1 FD1 (SEQ ID NO:39), obtained by removing 2 amino-acid residues from Wnt1 FD, Wnt2 FD1 (SEQ ID NO:40), obtained by removing 2 amino-acid residues from Wnt2 FD, Wnt2b FD1 (SEQ ID NO:41), obtained by removing 2 amino-acid residues from Wnt2b FD, Wnt3 FD1 (SEQ ID NO:42), obtained by removing 2 amino-acid residues from Wnt3 FD, Wnt4 FD1 (SEQ ID NO:43), obtained by removing 2 amino-acid residues from Wnt4 FD, Wn5a FD1 (SEQ ID NO:44), obtained by removing 2 amino-acid residues from Wnt5a FD, Wnt5b FD1 (SEQ ID NO:45), obtained by removing 2 amino-acid residues from Wnt5b FD, Wnt6 FD1 (SEQ ID NO:46), obtained by removing 2 amino-acid residues from Wnt6 FD, Wnt7a FD1 (SEQ ID NO:47), obtained by removing 2 amino-acid residues from Wnt7a FD, Wnt7b FD1 (SEQ ID NO:48), obtained by removing 2 amino-acid residues from Wnt7b FD, Wnt8a FD1 (SEQ ID NO:49), obtained by removing 2 amino-acid residues from Wnt8a FD, Wnt8b FD1 (SEQ ID NO:50), obtained by removing 2 amino-acid residues from Wnt8b FD, Wnt9a FD1 (SEQ ID NO:51), obtained by removing 2 amino-acid residues from Wnt9a FD, maintain functional similarity with Wnt and may be used for the activation of the FZD receptors, and for the treatment of pain.

In another embodiment, the compound N-Formyl-Met-Asp-Gly-Cys-Glu-Leu (FOXY-5), comprising amino-acids 332-337 of human Wnt5a, maintains functional similarity with Wnt, and may be used in pain treatment. Furthermore, the term “Wnt” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of any Wnt isoform.

The term “Hh” shall be construed to include natural and artificial polypeptides that maintain sequence or functional similarity, or both, to any member of human Hedgehog family of ligands, including Shh, Dhh and Ihh, as typically understood by those of ordinary skill in the art, regardless of whether the candidate protein is named “Hh” or not. Functional similarity to Hh shall be construed as the property of an agent to bind to, and inhibit a Patched (PTCH) receptor, to activate a Smoothened (SMO) receptor, and to promote neuronal differentiation. For example, a polypeptide selected from the group consisting of SHH_FD (SEQ ID NO:25) comprising amino-acids 24-197 of human Shh, DHH_FD (SEQ ID NO:26) comprising amino-acids 23-198 of human Dhh, and IHH_FD (SEQ ID NO:27) comprising amino-acids 28-202 of human Ihh, may be used to promote neuronal differentiation for the purpose of treating pain. These sequences are highly conserved, having at least 95% homology among animal species, including Homo sapiens, Rattus norvegicus, Mus musculus, Lipotes vexillifer, and Gallus gallus. Many other polypeptides derived from SHH_FD, DHH_FD or IHH_FD can be generated, which may maintain functional similarity with Hh, and may therefore be used in the treatment of pain. For example, the removal or exchange of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino-acid residues from Shh_FD, Dhh_FD or Ihh_FD, may generate polypeptides with functional similarity to Hh, which may be used in the treatment of pain. These examples suggest that polypeptides of any length that maintain at least 95% homology with Hh, may also maintain the ability to activate a SMO receptor, and may therefore be used in the treatment of pain. For example the polypeptides SHH_FD1 (SEQ ID NO:52), DHH_FD1 (SEQ ID NO:53), and IHH_FD1 (SEQ ID NO:54), obtained by removing 2 amino-acid residues from SHH_FD, DHH_FD, and IHH_FD, respectively, maintain functional similarity to Hh, and may be used to promote neuronal differentiation for the purpose of treating pain. Furthermore, the term “Hh” shall be construed to also include nucleic acid sequences, including DNA and RNA sequences equivalent to the amino-acid sequence of Hh.

The term “TrkA” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human TrkA receptor, as typically understood by those of ordinary skill in the art. For example the term “TrkA” shall be construed to include all isoforms and variants of the human TrkA protein.

The term “TrkB” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human TrkB receptor, as typically understood by those of ordinary skill in the art. For example the term “TrkB” shall be construed to include all isoforms and variants of the human TrkB protein.

The term “TrkC” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human TrkC receptor, as typically understood by those of ordinary skill in the art. For example the term “TrkC” shall be construed to include all isoforms and variants of the human TrkC protein.

The term “CNTF receptor” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human CNTF receptor, as typically understood by those of ordinary skill in the art. For example the term “CNTF receptor” shall be construed to include all isoforms and variants of the human CNTF receptor.

The term “Ret receptor” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human Ret receptor, as typically understood by those of ordinary skill in the art. For example the term “Ret receptor” shall be construed to include all isoforms and variants of the human Ret receptor.

The term “GDL receptor” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human GDNF receptor family, as typically understood by those of ordinary skill in the art. For example the term “GDL receptor” shall be construed to include all isoforms and variants of the human GDNF family receptor alpha-1, human GDNF family receptor alpha-2, human GDNF family receptor alpha-3, and human GDNF family receptor alpha-4.

The term “LIF receptor” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human LIF receptor alpha, as typically understood by those of ordinary skill in the art. For example the term “LIF receptor” shall be construed to include all isoforms and variants of the human LIF receptor.

The term “erythropoietin receptor” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human erythropoietin (EPO) receptor, as typically understood by those of ordinary skill in the art. For example the term “erythropoietin receptor” shall be construed to include all isoforms and variants of the human erythropoietin receptor.

The term “c-Met” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human c-Met receptor, as typically understood by those of ordinary skill in the art. For example the term “c-Met receptor” shall be construed to include all isoforms and variants of the human c-Met receptor.

The term “angiotensin receptor” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human angiotensin II receptor, as typically understood by those of ordinary skill in the art. For example the term “angiotensin receptor” shall be construed to include human angiotensin receptor type-1, human angiotensin receptor type-1b, human angiotensin receptor type-2, other types of angiotensin receptors, and all their human isoforms and splice variants.

The term “frizzled” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human frizzled receptor, as typically understood by those of ordinary skill in the art. For example the term “frizzled” shall be construed to include human Frizzled-1, human Frizzled-2, human Frizzled-3, human Frizzled-4, human Frizzled-5, human Frizzled-6, human Frizzled-6, human Frizzled-7, human Frizzled-8, human Frizzled-9, human Frizzled-10, other frizzled domain-containing proteins, and their splice variants in humans.

The term “Patched” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human Patched-1 receptor (PTCH), as typically understood by those of ordinary skill in the art. For example the term “Patched” shall be construed to include human Patched-1 isoform S, human Patched-1 isoform M, human Patched-1 isoform L, human Patched-1 isoform L′, human Patched-2 isoform 1, human Patched 2 isoform 2, other Patched domain-containing proteins, and their splice variants in humans.

The term “Smoothened” shall be construed as including proteins that maintain sequence or functional similarity, or both, to the human Smoothened (SMO) receptor, including its isoforms, as typically understood by those of ordinary skill in the art.

The terms “cell”, “cell line” and “cell culture” include progeny. It is understood that all progeny may not be precisely identical in DNA or protein content, due to deliberate or accidental mutations. Variant progeny that have the same function or biological property as determined in the originally characterized cell, are included. The cells used in the present invention are generally eukaryotic or prokaryotic cells.

The term “vector” shall be construed as meaning a DNA or RNA sequence which is functionally linked to a suitable polynucleotide control sequence capable of producing the expression of the DNA in a cell. Such control sequences include a promoter to initiate transcription, an optional operator sequence to control transcription, an origin of replication, a cloning site, selectable markers, a sequence encoding RNA ribosome binding sites, and sequences that control the termination of transcription and translation. The vector may be a plasmid, a phage or virus particle, a cosmid, an artificial chromosome, or a genomic insert. After introduction in a cell, the vector may replicate and function independently of the cell genome, or may in some cases integrate into the genome itself. In the present specification, “vector” and “plasmid” may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of vectors which serve equivalent function and which are known in the art.

Alternatively, a vector may include, in addition to the elements described above, an inducible promoter, which activates gene expression only under specific, controllable conditions. Such controllable conditions include a specific temperature (e.g. heat shock promoter), a specific chemical (e.g. doxycycline, dexamethasone, etc.), or other conditions.

The terms “transformation”, “transfection” and “infection” shall be construed as meaning the introduction of a vector containing a polynucleotide sequence of interest into a suitable cell, whether or not any coding sequences of that vector are expressed. The cell where the vector is introduced is termed “host cell”. The introduced polynucleotide sequence may be from the same species as the host cell, from a different species, or may be a hybrid polynucleotide sequence containing sequences from both the same species and a different species than the host cell. Methods of transfection include electroporation, calcium phosphate, liposome, DEAE-dextran, microinjection, polybrene, and others. The term “infection” shall be construed as meaning a transfection by use of a viral vector. Examples of viral vectors include adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), herpes simplex virus (HSV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV) and others.

In addition to the above definition, the term “transfection” shall be construed to also include the introduction of a protein into a host cell. Protein transfection may be achieved using a variety of commercially available reagents and kits, e.g. cationic lipid mixtures, peptides, etc.

The term “stem cell” shall be construed as including cells that maintain the ability to become any type of cell that is present in an organism. Examples of stem cells include embryonic stem cells, mesenchymal stem cells, amniotic stem cells, dental pulp stem cells, induced pluripotent stem cells (iPSCs), and others. The term “progenitor cell” is construed as including cells that maintain the capability of becoming a subset of all the cell types present in an organism. For example, a neural progenitor cell is a progenitor cell that can become one of several types of cells present in the nervous system. In the present specification, “stem cell” and “progenitor cell” may be used interchangeably, as the progenitor cell is a type of stem cell that has acquired some individual characteristics that differentiate it from a stem cell. For example, a neural progenitor cell may express a partially different subset of genes than a stem cell, which limit the ability of the neural progenitor cell to become only a cell type present in the nervous system. However, it is understood that both stem cells and progenitor cells are continuously dividing cells, and produce through division daughter cells identical to the dividing parent cell, over a large number of divisions. Alternatively, cells with stem cell properties may be derived from natural sources (e.g. cancer cells).

The term “pain” shall be construed to include the perception of pain, as commonly understood in medical practice and/or human communication. Pain symptoms may include acute or chronic pain, following an injury, neurodegenerative disease, cancer, diabetes, rheumatism, genetic variation, pain of unknown origin, etc. The pain threshold is a minimum value of the stimulus at which the stimulation is perceived as pain. The pain threshold does not have a specific value, and may vary in different people, or in the same person at different times. Stimuli that are considered normal for the average individual may become abnormal and cross above the pain threshold in specific individuals, or in the average individual under specific conditions. Pain sensitivity varies inversely to the pain threshold, for example a higher pain threshold is equivalent to a lower pain sensitivity.

The terms “pain level” and “pain intensity” shall be construed to represent a numerical or symbolic value assigned to pain through a measurement or evaluation method selected from a group which includes, but is not limited to a Wong-Baker FACES pain rating scale, a numeric pain rating scale, a pain quality assessment scale, a multidimensional pain questionnaire, a verbal descriptor scale, a visual analogue scale, the FLACC scale, the CRIES scale, the COMFORT scale, an MRI scan, a heart rate measurement, an electrocardiogram scan, a dolorimeter, an algesiometer, a von Frey filament scale, a measurement of withdrawal latency (in seconds) from radiant heat (Hargreaves method, the tail flick test, the tail withdrawal test, the hot plate test), ultrasound, lasers, etc. A multitude of subjective evaluations, physiological measurements, or combinations thereof can be used to determine pain level and variations thereof.

The term “pain sensitivity” shall be construed to represent the ability of a subject to perceive pain. Some subjects may perceive as painful stimuli which are not normally perceived as painful in an average subject. Such increase in pain sensitivity may be the result of an injury, disease, disorder, or genetic condition. Pain sensitivity varies inversely to the pain threshold.

The term “nociception” shall be construed to include the response of sensory neurons to external stimuli, including mechanical, thermal, and chemical stimuli, and the transmission of said response to the spinal cord. Because nociception typically results in a perception of pain, herein the terms “pain” and “nociception” are used interchangeably.

The term “flavone” shall be construed to represent the compound known as 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one) under IUPAC rules.

The Description of a New Mechanism of Pain

A new model of pain regulation is described, wherein pain is determined by the number of hyper-excitable immature neurons present in the spinal cord dorsal horns (Rusanescu et al., J Cell Mol Med. 19:2352-2364 (2015)). According to this model, an increase in the number of spinal cord dorsal horn immature neurons results in an increased pain and pain sensitivity, whereas a reduction of the number of spinal cord dorsal horn immature neurons results in a reduction of pain and pain sensitivity.

Spinal nerve injury is typically followed by a period of 2-3 months of chronic pain. In experimental animals subjected to unilateral chronic constriction injury of the sciatic nerve (CCI), this extended period of pain has been considered to be similar to human cases of chronic pain (Bennett et al., Pain 33, 87-107 (1988)), and the CCI method is therefore used in an experimental setting to simulate human pain.

In experimental mice and rats, CCI leads to an induction of adult neurogenesis (Rusanescu et al., J Cell Mol Med. 19, 2352-2364 (2015)). FIG. 1 depicts the increase in EdU staining, specific for proliferating cells, measured by immunofluorescence and quantified in the spinal cord ipsilateral to CCI, four weeks after CCI. Since EdU stains non-specifically all proliferating cells, a neuron-specific marker, nestin, is depicted in FIG. 2 as having a similar variation in time and ipsilateral location with EdU. Nestin is a marker specific for neural stem cells (Schechter et al., Stem Cells 25, 2277-2282 (2007)). The largest increase in nestin expression is reached only 6 weeks after CCI. The delayed increase in EdU and nestin staining, explained by the limited rate of adult neurogenesis, and by the time necessary for these neuron progenitors to migrate to the dorsal horn, is essential for the understanding of the extended symptoms of chronic pain.

Subsequent to nestin expression, the process of neuronal differentiation continues in neuron progenitor cells by transiting sequential stages of neuronal differentiation characterized by the expression of Mash1 (Bertrand et al., Nat Rev Neurosci. 3:517-30 (2002)), doublecortin (Brown et al., J Comp Neurol. 467:1-10 (2003)) and Notch3 (Rusanescu et al., J Cell Mol Med. 18:2103-2016 (2014)). FIG. 3, FIG. 4 and FIG. 5 show the gradual increase in Mash1, doublecortin and Notch3 expression, respectively, over six weeks after CCI. The increases in Mash1, doublecortin and Notch3 expression are significantly larger on the side ipsilateral to the injured nerve, indicating the increased presence of immature neurons.

The presence of neuron progenitor cells in the spinal cord has been demonstrated before, however the complete differentiation of said cells into mature functional neurons, which are integrated into spinal cord circuits, has never been demonstrated. FIG. 6 depicts a 21% increase in the number of neurons in the ipsilateral spinal cord dorsal horn layers I and II, relative to the contralateral side, six weeks after CCI. This excess of neurons on the ipsilateral side can be observed for up to 4 months after CCI (Rusanescu et al., J Cell Mol Med. 19:2352-2364 (20015)), which indicates that these neurons are integrated into the spinal cord circuits, otherwise these neurons would be unable to survive for several months. The integration of these newly generated neurons while in an immature and hyper-excitable state, into spinal cord neuronal circuits that process pain signals results in increased spinal cord excitability and a chronic perception of pain for as long as said neurons are in an immature state. In the case of CCI, the presence of immature neurons can last for several months, until such neurons mature and become less excitable. In the case of genetic conditions which prevent neuronal maturation (Rusanescu et al., J Cell Mol Med. 18:2103-2016 (2014)), or which constantly generate new immature neurons, increased spinal cord excitability and pain can be life-long conditions.

The representation of the minimal mechanical stimulus capable of inflicting pain after CCI (mechanical threshold) versus time on a semi-log graph (FIG. 7, A) demonstrates the presence of at least two distinct mechanisms responsible for pain. The arrows in FIG. 7 indicate an inflexion point at weeks 4-5, which indicates a change in the mechanism of pain. The early phase of pain (weeks 1-4) was due to an inflammatory mechanism triggered by the immune system (Ren et al., Nat Med. 16:1267-76 (2010)). By week 4 after CCI, the gliosis associated with the inflammatory response decreased to control levels (Rusanescu et al., J Cell Mol Med. 19:2352-2364 (20015)), indicating that inflammation is not a cause for pain after week 4. During weeks 4-8, equivalent to chronic (long-term) pain, the main cause of pain is the increase in the number of spinal cord immature neurons, as depicted in FIGS. 1-6. FIG. 7 (B) depicts the variation in sensitivity to heat-induced pain, which shows a similar inflexion point at week 4. The response to mechanical stimuli on the contralateral paw depicted in FIG. 7 (C) was less intense and the inflexion point was shifted to week 5, which reflects the smaller number of immature neurons that reach the contralateral spinal cord. The response to radiant heat on the contralateral paw depicted in FIG. 7 (D) was similar to Sham animals.

The idea that the immature neurons generated by adult neurogenesis contribute to pain was tested by demonstrating the effects of artificially increasing or decreasing neurogenesis on pain sensitivity (Rusanescu et al., J Cell Mol Med. 19:2352-2364 (20015)). The level of neurogenesis is regulated by EGF and FGF2 concentrations (Vescovi et al., Neuron 11:951-66 (1993)). FIG. 8 (A) depicts the changes in the expression of neuron progenitor markers Mash1, doublecortin and Notch3 after CCI, when experimental rats are treated with intrathecal injections of either control saline (CCI+Veh), or inhibitors of EGF and FGF2 signaling (CCI+INH), or EGF and FGF2 active growth factors (CCI+GF). All three markers undergo an increase after GF treatment and a decrease after treatment with inhibitors of EGF and FGF2 (INH). FIG. 8 (B-E) depicts changes in pain sensitivity occurring after the same treatments as in (A). FIG. 8 (B) depicts a decrease in mechanical pain sensitivity after treatment with INH, both in CCI-treated rats and in control rats. FIG. 8 (C) depicts a decrease in thermal pain sensitivity, after treatment with INH. FIG. 8 (D) depicts an increase in mechanical pain after treatment with GF. FIG. 8 (E) depicts an increase in thermal pain sensitivity after treatment with GF. Overall, FIG. 8 indicates that there is a strong correlation between the number of spinal cord immature neurons and the level of pain sensitivity.

A Method of Using Promoters of Neuronal Differentiation for the Purpose of Reducing Pain

A reduction of the number of spinal cord dorsal horn immature neurons, for the purpose of reducing pain, is achieved by treatment with a compound that induces or promotes the differentiation of highly excitable immature neurons into mature neurons with low excitability. Many of the mature neurons generated through said treatment may be inhibitory neurons, which may contribute to the overall effect of reducing pain.

Any type of compound that promotes neuronal differentiation may be used in the treatment of pain. This invention describes the use of protein families having the well-established property of inducing neuronal differentiation by binding to, and activating a specific receptor, as typically understood by those of ordinary skill in the art in the field of the invention. This invention also describes the use of other compounds which are effective in treating pain by inducing neuronal differentiation. These molecules typically induce neuronal differentiation by binding to a specific receptor and activating down-stream signaling pathways and the transcription of neuron-specific genes in a manner similar to the natural ligands.

The specific receptors described herein that have the ability to induce neuronal differentiation, and therefore may be targeted by ligands intended to treat pain, include but are not limited to TrkA, TrkB, TrkC, CNTF, GDNF family receptor alpha-1, GDNF family receptor alpha-2, GDNF family receptor alpha-3, GDNF family receptor alpha-4, Ret, LIF receptor, c-Met, angiotensin receptor, erythropoietin receptor, FZD, PTCH, and SMO. It is understood that some of these receptors use co-receptors to perform some or all of their physiological functions. This list is included as an example, and is not intended to limit the applicability and range of this invention, as other receptors or cell signaling pathways may also directly or indirectly promote neuronal differentiation and may therefore be used in treating pain.

The activation of the TrkB receptor induces a long-term reduction in pain and pain sensitivity, contrary to existing concepts known to those of ordinary skill in the art in the field of the invention. The activation of the TrkB receptor can be achieved using an agent (ligand) selected from a group comprising BDNF, and other TrkB ligands. For example, BDNF treatment of an animal subjected to CCI results in a long-term reduction of pain in treated animals (FIG. 9). The reduction in the expression of Mash1, DCX and Notch3 after BDNF treatment (FIG. 9, A) correlates with the reduction in mechanical pain susceptibility (increased pain threshold), depicted in FIG. 9 (B). The similarity of BDNF effects in CCI-treated and Sham-treated animals is an indication that BDNF reduces pain by inducing the differentiation of immature neurons, which are present in both CCI and Sham-treated animals. Said similarity between CCI and Sham-treated animals is an indication that the action of BDNF is not mediated through alternative mechanisms for pain, such as preventing neuronal death or reducing inflammation, because these processes are absent in Sham or control animals. BDNF treatment may increase long-term sensitivity to thermal pain by reducing the number of inhibitory neurons generated in the spinal cord (FIG. 9,C). The introduction of a 3-week delay in the administration of BDNF treatment after CCI allows the initial production of a large number of neuron precursor cells (depicted in FIGS. 1-5). BDNF administration induces a short-term, brief increase in pain, followed by a long-term reduction in pain and pain susceptibility (FIG. 9 (D)). A delayed BDNF treatment also reduced thermal pain susceptibility (FIG. 9 (E)), by increasing the number of inhibitory neurons in the spinal cord.

In a different embodiment, agents that activate the TrkB receptors also include chemical compounds (Obianyo et al., Biochim Biophys Acta 1834:2213-2218 (2013)). Some TrkB ligands have a flavone structure, for example the 7,8-dihidroxyflavone (DHF) (Jang et al., Proc Natl Acad Sci USA. 107: 2687-92 (2010)). FIG. 10 depicts the effects of DHF administration on neuronal differentiation and pain, which are very similar to the effects of BDNF. DHF promotes the differentiation of immature neurons and reduces the expression of Mash1, DCX and Notch3 (FIG. 10, A). Concurrently, DHF also reduces long-term mechanical pain susceptibility (FIG. 10, B), but not thermal pain susceptibility (FIG. 10, C). A 3-week delay in DHF administration after CCI produces a brief short-term increase, followed by an extended long-term decrease in mechanical and thermal pain susceptibility (FIG. 10, D,E), similar to BDNF. The similarity between the effects of DHF and the effects of BDNF on pain levels is an indication that DHF and BDNF act through the same cellular mechanism, by activating the TrkB receptors and inducing neuronal differentiation, but not through other mechanisms proposed in the scientific literature in the field of the invention.

In another embodiment, many other natural and synthetic agents with the property of activating TrkB are known, which may be used in treating pain. These include, but are not limited to: deoxygedunin, dihydro-deoxygedunin, alpha-dihydrogedunol (Jang et al., PLOS One 5:e11528 (2010)), 7,8,3′-trihydroxyflavone (Yu et al., Biochem Biophys Res Comm. 422:387-92 (2012)), 7,8,2′-trihydroxyflavone, 7,8,3′-tridydroxyflavone, 5,7,8-trihydroxyflavone, 7,3′-dihydroxyflavone, 5,7,8,2′-tetrahydroxyflavone, 3,7-dihydroxyflavone, 4′-dimethylamino-7,8-dihydroxyflavone, 4′-dimethylamino-8-hydroxy-7-methoxyflavone (Liu et al., J Med Chem. 53:8274-86 (2010)), N-acetyl-serotonin, N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (Shen et al., Proc Natl Acad Sci USA. 109:3540-45 (2012)), 5-oxo-L-prolil-L-histidyl-L-tryptophan-methyl ester (LM22A-1), 2-[2,7-bis[[(2-hydroxyethyl)amino]sulfonyl]-9H-fluoren-9-ylidene]-hydrazinecarboxamide (LM22A-2), N-[4-[2′[5-amino-4-cyano-1-(2-hydroxyethyl)-1H-pyrazol-3-yl]-2-cyanoethenyl]phenyl]-acetamide (LM22A-3), and N,N′,N″-tris(2-hydroxyethyl)-1,3,5-benzenetricarboxamide (LM22A-4) (Massa et al., J Clin Invest. 120:1774-85 (2010)). Many other compounds derived by substitution from flavone, flavanone (2,3-dihydroflavone; 2-phenyl-4-chromanone), flavanonol (3-hydroxy-2,3-dihydro-2-phenylchromen-4-one, 3-hydroxyflavanone), flavanol (2-phenyl-3,4-dihydro-2H-chromen-3-ol), or chalcone (1,3-diphenyl-1-propen-3-one) may have the ability to activate the TrkB receptor and induce neuronal differentiation, and may therefore be used to treat pain.

In another embodiment, many other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate TrkB, directly or indirectly, and which as a result may be used in promoting neuronal differentiation and in treating pain.

In addition to polypeptides identical or similar to NGF, other agents with the property of activating TrkA are known, which may be used in treating pain. These include, but are not limited to gambogic amide. In addition to these examples, many other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate TrkA, directly or indirectly, and which as a result may be used in promoting neuronal differentiation and in treating pain.

In addition to polypeptides identical or similar to NT3, other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate TrkC, directly or indirectly, and which may as a result be used in promoting neuronal differentiation and in treating pain.

In addition to polypeptides identical or similar to CNTF, other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate the CNTF receptor, directly or indirectly, and which may be used in promoting neuronal differentiation and in treating pain.

In addition to polypeptides identical or similar to GDL, other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate Ret, directly or indirectly, and which may be used in promoting neuronal differentiation and in treating pain.

In addition to polypeptides identical or similar to LIF, other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate the LIF receptor, directly or indirectly, and which may be used in promoting neuronal differentiation and in treating pain.

In addition to polypeptides identical or similar to angiotensin, other agents with the property of being ligands for the angiotensin receptors are known, which may be used in treating pain. For example, 5,7-dimethyl-2-ethyl-3-[[4-[2(N-butyloxycarbonyl sulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazo[4,5-b]-pyridine (L-162,313) is a ligand for the angiotensin receptor type-1, and N(alpha)-nicotinoyl-Tyr-(N(alpha)-carboxybenzoyl-arginine)-Lys-His-Pro-Ile (GCP-42112A) is a ligand for angiotensin receptor type-2.

In addition to polypeptides identical or similar to EPO, other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate EPO receptors, directly or indirectly, and which may be used in promoting neuronal differentiation and in treating pain. For example, Epotris, a polypeptide comprising amino-acids 92-111 of human EPO, can induce neurite growth (Pankratova et al., Brain 133:2281-94 (2010)), which suggests that it may be used in treating pain.

In addition to polypeptides identical or similar to HGF, other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate c-Met receptors, directly or indirectly, and which may be used in promoting neuronal differentiation and in treating pain. For example, N-hexanoic-Tyr-Ile-(6)aminohexanoic amide (Dihexa) is a compound that binds to HGF and potentiates its ligand activity, inducing neuronal differentiation, and may therefore be used in treating pain.

In addition to polypeptides identical or similar to Hh, other agents with the ability to activate the SMO receptor and the Hh signaling pathway are known, which may be used in treating pain. For example, N-methyl-N′-(3-pyridinylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohehane (SAG) (Bragina et al., Neurosci Lett. 482:81-5 (2010)), and 2-(1-naphthoxy-6-(4-morpholinoanilino)-9-cyclohexylpurine (Wu et al., Chem Biol. 11:1229-1238 (2004)) are ligands for SMO, activate the Hh pathway and may promote neuronal differentiation, therefore may be used in the treatment of pain. In addition to these examples, many other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate SMO directly or indirectly, and which may be used in promoting neuronal differentiation and in treating pain.

In addition to polypeptides identical or similar to Wnt, other natural or synthetic compounds are known, or may be derived through chemical reactions from known compounds, which may have the ability to activate FZD receptors, directly or indirectly, and which may be used in promoting neuronal differentiation and in treating pain.

A limited number of individual flavones have been empirically known to have analgesic effects, which include flavone (Thirugnanasambantham et al., Clin Exp Pharmacol Physiol. 20:59-63 (1993)), procyanidin, rutin, hyperoside (Rylski et al., Acta Physiol Pol. 30:385-8 (1979)), isoliquiritigenin (Shi et al., Phytother Res. 26:1410-7 (2012)), gossypin (Viswanathan et al., Eur J Pharmacol. 98:289-91 (1984)), 3,3′-dihydroxyflavone, 5,6-dihydroxyflavone, 3,7-dihydroxyflavone, 6,3′-dihydroxyflavone (Vidyalakshmi et al., Pharmacol Biochem Behav. 96:1-6 (2010)), 7,2′-dimethoxy flavone, 7,3′-dimethoxyflavone, 7,4′-dimethoxyflavone, 7,8-dimethoxy flavone (Pandurangan et al., Eur J Pharmacol. 727:148-57 (2014)), 0-(beta-hydroxyethyl rutoside (Ramaswamy et al., Indian J Exp Biol. 23:219-20 (1985)), 5-hydroxyflavone, 7-hydroxyflavone, 2′-hydroxyflavone, 5,7-dihydroxyflavone (Thirugnanasambantham et al., J Ethnopharmacol. 28:207-14 (1990)), 3,6-dihydroxyflavone, 3,2′-dihydroxyflavone, 3,4′-dihydroxyflavone, 6,7-dihydroxyflavone, 3′,4′-dihydroxyflavone, 7,2′-dihydroxyflavone (Girija et al., Indian J Exp Biol. 40:1314-6 (2002)), myricetin, myricitrin, linarin, baicalin, baicalein, luteolin, hesperidin, and wogonin. The analgesic action of said flavones has been thought to occur by activating opioid or GABA receptors (Viswanathan et al., Eur J Pharmacol. 98:289-91 (1984); Girija et al., Indian J Exp Biol. 40:1314-6 (2002)), through anti-inflammatory action (Burnett et al., J Med Food. 10:442-451 (2007), or through the modulation of ion channels (Hagenacker et al., Eur J Pain. 14:992-8 (2010)). These actions activate cellular mechanisms that are distinct from the activation of TrkB and the induction of neuronal differentiation. The ability of said compounds to induce TrkB activation and neuronal differentiation in relation with their analgesic properties was not tested, therefore said compounds do not constitute prior art for the purpose of identifying new related compounds with analgesic properties based on their neuronal differentiation ability. In fact, until the date of this application, TrkB agonists have been universally considered to have the opposite effect, of promoting pain.

Methods for Screening and Identifying Compounds which can Reduce Pain

The idea of this invention, that agents with the ability to promote neuronal differentiation can also reduce pain, can be used to screen for, and identify new candidate treatments for pain.

Because immature neurons contribute to the perception of pain, new candidate agents for treating pain may be identified by screening libraries of chemical compounds for the ability to induce neuronal differentiation. Many methods for testing the ability of a compound to induce neuronal differentiation may be used, which are well-known to those of ordinary skill in the art in the field of the invention. Examples of screening methods include, but are not limited to: the measurement of neurite growth, immunofluorescence measurement of neuronal marker expression, binding of labeled ligands to neuron-specific receptors, secretion of neuron-specific molecules, changes in cell morphology, and other methods.

Chemical compounds identified as candidate agents for pain treatment by screening for neuronal differentiation may be further tested in animals, developed by chemical modification to generate other candidate compounds, or developed by pharmacological formulation for various routes of administration.

Pharmaceutical Formulation

Therapeutic formulations of the agents described herein as promoters of neuronal differentiation may be prepared for the purpose of administration to an individual as injections, perfusions, patches, by mouth (tablets, capsules, solutions, suspensions), by inhalation, by nanoparticles, by infusion pumps, as viral particles or as cell transplants.

Therapeutic formulations of the agents described herein as promoters of neuronal differentiation may be prepared for storage or administration as lyophilized formulations, aqueous solutions, powders, tablets, capsules, or plasmids, by mixing the purified agent with optional carriers, excipients or stabilizers commonly used in the art, all of which are termed “excipients”. Excipients include buffers, stabilizing agents, anti-oxidants, preservatives, detergents, salts, and other additives. Such additives must be nontoxic to cells or recipients at the dosages and concentrations used.

In another embodiment, therapeutic formulations may include cells or viruses modified to express the polypeptides described herein as promoters of neuronal differentiation.

Buffering agents maintain the pH of the agent formulation in a range which approximates physiological conditions. Suitable buffering agents for use with the current invention include organic and inorganic acids and salts thereof, such as citrate buffers (e.g. monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, etc.), succinate buffers (e.g. succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g. fumaric acid-sodium hydroxide mixture, fumaric acid-disodium fumarate mixture, etc.), gluconate buffers (e.g. gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, etc.), acetate buffers (e.g. acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.), phosphate buffers (e.g. monosodium phosphate-disodium phosphate mixture, etc.), trimethylamine salts (e.g. Tris), and other buffers.

Preservatives may be used to inhibit microbial growth in the formulation. Suitable preservatives for use with the current invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzalkonium halides, catechol, resorcinol, cyclohexanol, and others typically used in the art.

Stabilizers may be used to increase solubility, provide isotonicity, prevent denaturation, or prevent adherence to container of the agent. Suitable stabilizers for use with the current invention include polyhydric alcohols and sugars (e.g. glycerin, polyethylene-glycol, erythritol, xylitol, mannitol, sorbitol, inositol, trehalose, lactose, etc.), amino-acids (e.g. arginine, glycine, histidine, polypeptides, etc.), proteins (e.g. albumin, gelatin, etc), reducing agents (e.g. urea, glutathione, thioglycerol, sodium thioglycolate, sodium thiosulfate, etc.), and others commonly used in the art.

Detergents may be used to increase solubility and prevent aggregation of the formulation. Suitable detergents for use with the current invention include polysorbates (e.g. 20, 80, etc.), polyoxyethylene sorbitan ethers (TWEEN-20, TWEEN-80), polyoxamers and others commonly used in the art.

The formulations for in-vivo use must be sterile. This can be achieved by filtration through sterile filtration membranes.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture is provided, containing materials useful for the treatment of pain, as described in the invention. The article of manufacture comprises a label and a container. Suitable containers include vials, bottles, syringes, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. The container holds a composition which is effective in treating pain or in modifying cells used to treat pain. The active component in the composition is the agent, in the form of a polypeptide, chemical compound or vector. The label attached to the container indicates that the composition is used to treat pain. The article of manufacture may further include a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, dextrose solution or Ringer's solution. The article of manufacture may further include a third container comprising a pharmaceutically acceptable cell transfection system (e.g. liposomes, etc.). The article of manufacture may further include other materials necessary for the user, including other buffers, antibiotics, filters, syringes, and instructions for use.

Therapeutic Uses of Agents that Promote Neuronal Differentiation

It is intended that the agents described in the current invention may be used to treat a human or an animal. In one embodiment, the agent may be administered to a human or to an animal to treat pain. The present invention is directed to promote the differentiation of neurons in order to reduce their excitability and to reduce pain and pain sensitivity.

In another embodiment, the agent may be administered to treat chronic pain, that lasts for more than 3 months.

In another embodiment, the agent may be administered to treat medium term pain, that lasts between 3 weeks-3 months.

In another embodiment, the agent may be administered directly to the spinal cord as an epidural or intradural (intrathecal, spinal) injection. The injection may be repeated as needed, or may be administered by catheter, infusion pump, or both, over a period of time.

In another embodiment, the agent may be administered by mouth in the form of tablets, capsules, solutions or suspensions, until the elimination of pain is achieved, usually over a period of 3-5 weeks.

In another embodiment, if no pain symptom improvement is achieved within 2 weeks, the agent may be replaced with another agent with ligand properties for a different receptor family, as disclosed herein. Alternatively, two or more agents acting as ligands on different receptor families described herein may be administered simultaneously to treat pain.

The administration route for the agent shall be selected with the purpose of obtaining the optimal therapeutic concentration for the optimal period of time adequate for each individual. Concentration increases over the optimal therapeutic dose and duration shall be avoided in order to prevent adverse effects.

In another embodiment, proliferating host cells, including stem cells or progenitor cells, may be extracted from the same individual (autologous), or from another individual of the same species, or from a different species (heterologous). The agent may be introduced in these host cells, or in artificially modified cell lines, in the form of a polynucleotide (cDNA) that has the ability to generate the agent in the form of a polypeptide by transcription in-vivo, inside the host cell. The host cells that express the agent polypeptide may be introduced back into the same or into a different human host by transplantation, where the host cells are intended to secrete the polypeptide for therapeutic purposes.

In another embodiment, the transcription of the polynucleotide that has the ability to generate the agent in the form of a polypeptide may be placed under the control of an inducible promoter, for example containing TetO. Such promoters may be subjected to repeated cycles of induction and inhibition, as needed for pain treatment.

In cases where host cells expressing the agent are transplanted into a receiving individual different from the original individual donor of the host cells, the receiving individual may be administered immune suppression therapy in order to avoid the rejection of transplanted cells or tissues.

In a different embodiment, the nucleic acid sequence that expresses the agent may be administered in the form of a viral vector, or in other forms of gene therapy, to cells in a human for the purpose of becoming intracellular, expressing the agent and inducing neuronal differentiation. For example, vectors expressing BDNF polynucleotide may be introduced in the spinal cord in order to promote the differentiation of neuron progenitors and immature neurons. Viral vectors that may be used for agent polynucleotide delivery to cells inside a human include adenoviruses, adeno-associated viruses, retroviruses, and other types of viruses. Transfecting agents, encapsulation in liposomes, microparticles, nanoparticles, microcapsules, or administration in linkage to a ligand subject to receptor-mediated endocytosis may be also used to introduce agent nucleic acid sequences into cells, inside or outside a human. Alternatively, nucleic acid-ligand complexes can be formed, in which the ligand comprises a fusogenic peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. Alternatively, the nucleic acid may be targeted for in vivo cell specific uptake and expression, by targeting a specific receptor.

In a different embodiment, the host cells may be stimulated to produce their own agent, for the purpose of inducing neuronal differentiation. This procedure may be done using inducers of transcription specific for each agent, or by inducing agent-specific proteases which cleave the precursor protein into the biologically active “mature” polypeptide agent. This procedure may be performed on cells in cell culture or directly in cells inside a human.

In a different embodiment, cells induced to express the agent, whether by introducing a vector containing a polynucleotide that expresses the agent, or by directly introducing the agent polypeptide, may be introduced in an experimental animal for pre-clinical studies designed to develop a treatment for a disease or disorder. Alternatively, agent polynucleotides or polypeptides may be introduced directly into an experimental animal, using any methods well known to those of ordinary skill in the art.

EXAMPLES Example 1 Pain Sensitivity is Regulated by the Number of Spinal Cord Immature Neurons

The increase in the number of spinal cord immature neurons typically induces an increase in pain sensitivity. This idea was tested in commercially available Notch ko mice, which were analyzed for both pain sensitivity and the number of spinal cord immature neurons. Five Notch3 ko mice and five wild-type B6 mice, all male and 3 months old, were sacrificed by perfusion-fixation with 4% paraformaldehide. The fixed spinal cords were removed, then were frozen in Tek-OCT, then sliced in a microtome. The 35 micrometer-thick spinal cord slices were blocked in 2% bovine serum albumin in PBS, then double stained for immature neuron marker Calretinin and for mature neuron marker NeuN, using secondary antibodies with different absorption wavelengths. The slices were visualized by immunofluorescence, and the numbers of Calretinin-positive and NeuN-positive cells in the spinal cord layers I-II, responsible for pain transmission, were determined. Statistical analysis was performed using 5 slices from each animal. In Notch3 ko mice, the number of mature neurons showed an 86% decrease, and the number of immature neurons showed a 214% increase relative to wild-type mice. Concurrently, 10 Notch3 ko mice and 10 wild-type mice were tested for mechanical pain sensitivity using a series of von-Frey filaments. Mechanical pain was tested weekly for 4 weeks. The pain threshold in wild-type mice was 1-1.5 grams, while in Notch3 ko mice the pain threshold was 0.03-0.06 grams, indicating that pain sensitivity was 17-50 fold higher in Notch3 ko mice. This example shows a parallel between pain sensitivity and the number of immature neurons.

Example 2 Chronic Constriction Injury of the Sciatic Nerve Induces a Simultaneous Increase in the Number of Spinal Cord Immature Neurons, and in Pain Sensitivity

The idea that immature spinal cord neurons can contribute to pain was tested using a standard experimental animal model, the chronic constriction injury of the sciatic nerve (CCI). Fifteen two-month old rats were subjected to CCI under pentobarbital anesthesia. Three chromic gut ligature were tightly placed around the right sciatic nerve. Fifteen control rats were subjected to the same surgery, but without placing the ligatures (Sham). Mechanical pain sensitivity on the hind paws was tested weekly, using a set of calibrated von-Frey filaments applied to the plantar surface. Thermal pain sensitivity on the plantar surface was tested weekly by applying a beam of radiant heat. The results are shown in FIG. 7, for the ipsilateral paw (A, B) and for the contralateral paw (C, D). The side ipsilateral to the injured nerve shows a much larger increase in pain sensitivity relative to the contralateral side. At weeks 2, 4 and 6 after CCI, groups of three rats were sacrificed by perfusion-fixation under anesthesia, the spinal cords were removed, frozen, and sliced in 35 micrometer-thick slices. The slices were blocked in a 5% solution of bovine serum albumin, then incubated with primary antibodies for immature neuron markers nestin, Mash1, DCX and Notch3. The slices were then incubated with dye-linked secondary antibodies and imaged by immunofluorescence. The immunofluorescence images were quantified for marker expression in the ipsilateral and contralateral halves of the gray matter, using NIH-ImageJ. The quantification of nestin (FIG. 2), Mash1 (FIG. 3), DCX (FIG. 4) and Notch3 (FIG. 5) is shown. The increased expression of all 4 markers in the ipsilateral spinal cord after CCI indicates a larger number of immature neurons, and correlates with the increased pain sensitivity. The timing of the largest increase in the number of immature neurons at 6 weeks coincides with the timing of the second phase of pain sensitivity, indicating that long-term pain may be the result of immature neuron activity.

Example 3 A Reduction in the Number of Spinal Cord Immature Neurons Results in Reduced Pain Sensitivity

The epidermal growth factor (EGF) and the fibroblast growth factor 2 (FGF2) are known to be necessary for neurogenesis and the production of new neurons. Therefore, a reduction in the number of immature neurons present in the spinal cord was achieved by reducing the effective intraspinal activity of FGF2 and EGF. FGF2 concentration was reduced using a FGF2-specific neutralizing antibody. EGF signaling was inhibited using erlotinib, an inhibitor of the EGF receptor. The FGF2 antibody (10 microliters, 0.2 miligrams/ml) and erlotinib (5 microliters, 1 miligram/ml) were injected together (INH, FIG. 8) into the spinal canal (intrathecal injection) every other day for the first 3 weeks after CCI, using a phosphate buffer saline (PBS) vehicle. Control rats were injected with PBS vehicle alone (Veh). INH and Veh were each injected in separate rat groups, both in Sham animals (Sham+INH and Sham+Veh) and in CCI animals (CCI+INH and CCI+Veh). Each treatment group consisted of 9 rats. The rats were tested weekly for nociceptive sensitivity to both mechanical (Von Frey method) and thermal (Hargreaves method) stimuli, over a period of 8 weeks. INH treatment reduced mechanical nociceptive sensitivity in both CCI and Sham rats (FIG. 8, B). INH treatment also reduced thermal nociceptive sensitivity in CCI rats (FIG. 8, C). At week 4 after CCI, 3 animals from each treatment group were sacrificed by perfusion-fixation, their spinal cords were harvested, frozen and sliced on a microtome into 35-micrometer thick slices. The slices were blocked in a solution of bovine serum albumin and Tween-20 in PBS, then stained with primary antibodies specific for immature neuron markers Mash1, DCX and Notch3, and with dye-linked secondary antibodies, then imaged by immunofluorescence. The expression of each marker was individually quantified by immunofluorescence in the ipsilateral and contralateral halves of the spinal cord gray matter. Statistical analysis was performed using 5 slices from each spinal cord. FIG. 8 (A) shows that the INH treatment prevented the CCI-induced increase normally observed in the expression of all three markers. This example indicates that a reduction in the number of spinal cord immature neurons results in a reduction of pain sensitivity.

Example 4 An Increase in the Number of Spinal Cord Immature Neurons Induces Increased Pain Sensitivity

This example details an experiment opposite to Example 3. In order to stimulate spinal cord neurogenesis and increase the number of immature spinal cord neurons, recombinant FGF2 (10 microliters, 0.1 micrograms/ml) and EGF (5 microliters, 0.1 microgram/ml) were injected together (GF, FIG. 8) into the spinal canal (intrathecal injection) every other day for the first 3 weeks after CCI, using a phosphate buffer saline (PBS) vehicle. Control rats were injected with PBS vehicle alone (Veh). GF and Veh were each injected both in Sham animals (Sham+GF and Sham+Veh) and in CCI (CCI+GF and CCI+Veh) animals. Each treatment group consisted of 9 rats. The rats were tested weekly for nociceptive sensitivity to both mechanical (von Frey method) and thermal (Hargreaves method) stimuli, over a period of 8 weeks. GF treatment increased mechanical nociceptive sensitivity in both CCI and Sham rats (FIG. 8, D). GF treatment also increased short term thermal nociceptive sensitivity in both CCI and Sham rats (FIG. 8, E). At week 4 after CCI, 3 animals from each treatment group were sacrificed by perfusion-fixation, their spinal cords were harvested, frozen and sliced on a microtome into 35-micrometer thick slices. The slices were blocked in a solution of bovine serum albumin and Tween-20 in PBS, then stained with primary antibodies specific for immature neuron markers Mash1, DCX and Notch3, and with dye-linked secondary antibodies, then imaged by immunofluorescence. The expression of each marker was individually quantified by immunofluorescence in the ipsilateral and contralateral halves of the spinal cord gray matter. Statistical analysis was performed using 5 slices from each animal. FIG. 8 (A) depicts the increase in the expression of Mash1, DCX and Notch3 over the CCI-induced increase normally observed, caused by GF treatment. This example indicates that an increase in the number of spinal cord immature neurons results in an amplification of pain sensitivity.

Example 5 BDNF Concurrently Reduces the Number of Spinal Cord Immature Neurons and Pain Sensitivity.

TrkB is a classical example of receptor that induces neuronal differentiation. At the same time, TrkB activation has been known to induce pain. Therefore, FIG. 9 depicts the testing of BDNF both as a promoter of neuronal differentiation and as a regulator of pain susceptibility. Two groups of 9 rats were subjected to CCI and two groups of 9 rats to Sham surgery. Immediately after surgery, BDNF (10 microliters, 5 microgram/ml) was injected in the spinal canal (intrathecally) every other day for 3 weeks, to one group of CCI (CCI+BDNF) and one group of Sham (Sham+BDNF) rats. The other 2 groups of rats were injected with Vehicle (PBS) alone (CCI+Veh and Sham+Veh). After 4 weeks, 3 rats in each group were sacrificed by perfusion-fixation, the spinal cords were collected and sliced into 35 micrometer-thick slices. The slices were blocked in 2% BSA, then stained with primary antibodies for Mash1, DCX and Notch3, and with a dye-linked secondary antibody. The slices were imaged by immunofluorescence microscopy, and the stained areas were quantified for the ipsilateral and contralateral halves of the gray matter, using NIH-ImageJ. Statistical analysis was performed using 5 slices from each rat. FIG. 9 (A) depicts the effect of BDNF as a promoter of neuronal differentiation, by reducing the expression of all three markers. BDNF induced the immature neurons that express Mash1, DCX and Notch3 to undergo accelerated differentiation, which is expected to result in a reduced excitability. The same groups of rats were concurrently tested for pain sensitivity, using the von Frey method for mechanical pain and the Hargreaves method for thermal pain. FIG. 9 (B) depicts the long-term increase in mechanical pain threshold (decrease in pain sensitivity) caused by BDNF in both CCI and Sham rats. Although the BDNF treatment was applied for only 3 weeks, the decrease in pain lasted for 3 months because the immature neurons with high excitability, which regulate pain levels, are regenerated at a slow rate. The BDNF treatment increased long-term thermal pain sensitivity (FIG. 9, C) probably by reducing the number of inhibitory neurons involved in thermal pain transmission.

Example 6

Delayed BDNF Treatment after CCI Produces a Brief, Short-Term Increase in Pain Sensitivity, Followed by a Long-Term Decrease in Pain

The same experiments detailed in Example 5 were repeated, with the only modification that the BDNF treatment was delayed for 3 weeks after CCI. In this case, BDNF administration first induced a 2-3 day increase in both mechanical and thermal pain sensitivity, followed by a long-term decrease in pain sensitivity (FIG. 9, D,E). The 3 week delay in BDNF administration allowed an initial accumulation of neural progenitor cells, as depicted in FIGS. 1-4. Upon BDNF administration, all the neural stem cells present were induced to differentiate synchronously, first generating a large number of highly excitable immature neurons, which were responsible for the initial brief increase in pain sensitivity. These immature neurons continued to mature into low-excitable mature neurons, which were responsible for the long-term decrease in pain sensitivity (increased pain threshold to pain). This example confirmed that immature neurons are responsible for the regulation of pain, and explains why BDNF has been considered to be an inducer of pain.

Example 7 DHF Concurrently Reduces the Number of Spinal Cord Immature Neurons and Pain Sensitivity.

The same experiments described in Example 5 were repeated with the modification that DHF was used instead of BDNF as a TrkB ligand. FIG. 10 depicts the testing of DHF both as a promoter of neuronal differentiation and as a regulator of pain. Two groups of 9 rats were subjected to CCI and two groups of 9 rats to Sham surgery. Immediately after surgery, DHF (1 ml, 70 miligram/ml) was injected intraperitoneally every other day for 3 weeks, to one group of CCI (CCI+DHF) and one group of Sham (Sham+DHF) rats. The other 2 groups of rats were injected with Vehicle (PBS) alone (CCI+Veh and Sham+Veh). After 4 weeks, 3 rats in each group were sacrificed by perfusion-fixation, the spinal cords were collected and sliced into 35 micrometer-thick slices. The slices were blocked in 2% BSA, then stained with primary antibodies for Mash1, DCX and Notch3, and with a dye-linked secondary antibody. The slices were imaged by immunofluorescence microscopy, and the stained areas were quantified for the ipsilateral and contralateral halves of the gray matter, using NIH-ImageJ. Statistical analysis was performed using 5 slices from each rat. FIG. 10 (A) depicts the effect of DHF as a promoter of neuronal differentiation, by reducing the expression of all three markers. The same groups of rats were concurrently tested for pain sensitivity, using the von Frey method for mechanical pain and the Hargreaves method for thermal pain. FIG. 10 (B) depicts the long-term increase in mechanical pain threshold (decrease in pain sensitivity) caused by DHF in both CCI and Sham rats. Although the DHF treatment was applied for only 3 weeks, the decrease in pain lasted for 3 months. The DHF treatment increased long-term thermal pain sensitivity (FIG. 9, C) probably by reducing the production of inhibitory neurons involved in thermal pain transmission.

Example 8

Delayed DHF Treatment after CCI Produces a Brief Short-Term Increase in Pain Sensitivity, Followed by a Long-Term Decrease in Pain

The same experiments detailed in Example 7 were repeated, with the only modification that the DHF treatment was delayed for 3 weeks after CCI. In this case, DHF administration first induced a 2-3 days increase in both mechanical and thermal pain sensitivity, followed by a long-term decrease in pain sensitivity (FIG. 10, D,E). The 3 week delay in BDNF administration allowed an initial accumulation of neural progenitor cells, as depicted in FIGS. 1-4. Upon DHF administration, all the neural stem cells present were induced to differentiate synchronously, first generating a large number of highly excitable immature neurons, which were responsible for the initial brief increase in pain sensitivity. These immature neurons continued to mature into low-excitable mature neurons, which were responsible for the long-term decrease in pain sensitivity (increased pain threshold to pain). This example confirmed that DHF can be used in an animal to treat pain.

Example 9 Use of Dihexa to Treat Pain in a Human

Dihexa (N-hexanoic-Tyr-Ile-(6)aminohexanoic amide), an activator of c-Met receptor, is administered orally, 200 miligrams/day for 4 weeks, to a human for the purpose of treating pain. The subject is having chronic pain for the previous 6 months, as a result of injury. Before the beginning of treatment, and weekly thereafter for 6 months, the subject is tested for pain levels using a combination of subjective (numeric rating scale) and physiological (dolorimetric) evaluation methods. After 2 weeks of treatment the subject may report decreased pain. After 4 weeks of treatment, the subject may report that pain sensitivity is back to normal levels. Increased pain sensitivity may reoccur after 3 months, and then the treatment may be repeated.

Example 10 Combined Use of NT4 and a Flavone to Treat Pain in a Human

NT4 is a TrkB ligand and inducer of neuronal differentiation that may be used to treat pain. NT4 (200 microliters, 1 microgram/ml) is injected every other day for 3 weeks (10 injections total) into the spinal canal of a human patient diagnosed with fibromyalgia. The patient may report increased pain after 4-5 days, but the increase in pain subsides after 7-10 days with continued treatment. The patient may report decreased pain after 2 weeks, and no pain after 3-4 weeks. To prevent pain reoccurrence, a maintenance dose of 7,8-dihydroxy-4′-dimethylaminoflavone-8-O-glucozide (3-4 grams per day for 4-5 consecutive days) may be administered every month.

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1. A screening method for the identification, optimization, development and selection of agents having the ability to treat pain in an individual, comprising of assays that analyze and optimize the ability of these agents to induce or promote neuronal differentiation.
 2. The method of claim 1, wherein the ability of an agent to treat pain is measured in a human or in a mammal by administering that agent, alone or in combination with other agents, in an amount sufficient to cause a detectable decrease in pain, as determined by a pain or nociception measurement method.
 3. The method of claim 1, wherein neuronal differentiation is measured as a change in cellular properties selected from the group consisting of anatomical, electrophysiological, gene expression, RNA expression, protein expression, chemical, biochemical, immunofluorescence, immunochemical, immunological, physiological, spectroscopic, caloric, optic, metabolic, and secretory properties of a cell, measured in cell culture or in a mammal.
 4. The method of claim 1, wherein the agent is an agonist, antagonist, partial agonist or inverse agonist of a receptor selected from the group consisting of TrkA, TrkB, TrkC, p75trk, a CNTF receptor, an EGF receptor, a FGF receptor, GDNF family receptor alpha1, GDNF family receptor alpha2, GDNF family receptor alpha3, GDNF family receptor alpha4, a RET receptor, a LIF receptor, an angiotensin receptor, c-Met, a Frizzled receptor (FZD) and a Smoothened receptor.
 5. The method of claim 4, wherein the agent is a chemical compound, an antibody or a peptide comprising between 2 and 400 amino-acid residues.
 6. The method of claim 5, wherein the peptide has 95%-100% homology to a polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.
 7. The method of claim 1, wherein the agent is selected from a group or library containing between 1 and 1 billion chemical compounds.
 8. The method of claim 1, wherein the agent is an RNA molecule comprising between 8 and 60 nucleotides
 9. The method of claim 1, wherein the agent is selected from the endogenous genes in a human.
 10. The method of claim 9, wherein the endogenous gene is artificially modified by gene editing methods. 